THE ACTION OF OXIDIZING AGENTS ON WOOL KERATIN AND RELATED PROTEINS.

A thesis submitted to

The University of New South Wales

for the degree

of

Doctor of Philosophy

by

Morris Burke

,- ...... ,,.- ,'-"' ., ~, ...., t" 1 ~ K.;:N51NC]TQ \. School of Textile Tee

The University of New South Wales. 1966 This is to certify that the work described in this thesis was done by me in the School of Textile Technology, The University of New South Wales, and has not been submitted previously for any other university degree or award. ACKNOWLEOOEMENTS

The author wishes to acknowledge with thanks the assistance and advice given in this work by Associate Professor C. H. Nicholls.

The author is also grateful to the Wool Research Comnittee for the provision of a Wool Research Fellowship and funds for equipment which enabled this work to be carried out. CONTENTS Page CHAPTER I. INTRODUCTION 1 The Chemical Nature of Oxidation 2

The Nature of Oxidizing Agents 7 The Chemical Nature of Proteins 10 The Oxidation of Amino Acids and Proteins 22

(1) The oxidation of cystine 24 (ii) The oxidation of tyrosine 31 (111) The oxidation of tryptophan 34

(iv) The oxidation of the other amino acid residues 36 (v) Conclusions 36

CHAPTER II. THE OXIDATION OF AMINO ACIDS AND PROTEINS AS STUDIED BY POTENTIOMETRY 39 The Theoretical Principles of the Method 39 Application of the method for investigating the oxidation of amino acids 41 Experimental procedure 42 The Oxidation of the Amino Acids 44 (1) The oxidation of cyetine 44 (a) Oxidation by potassium permanganate 44 (b) Oxidation by chlorine 50 (11) The oxidation of cysteine 51 (a) Oxidation by potassium permanganate 51 (b) Oxidation by chlorine 53 Page (111) The oxidation of methionine 63 (a) Oxidation by potassium permanganate 63 (b) Oxidation by chlorine 54 (iv) The oxidation of tyrosine 54

(a) Oxidation by potassium permanganate 54 (b) Oxidation by chlorine 66 (v)' The oxidation ot tryptophan 67 (a) Oxidation by potassium permanganate 67 (b) Oxidation by chlorine 58 The Oxidation ot The Amino Acids in The Presence of Sodium Chloride by Acidified Potassium Permanganate 69 (1) The oxidation ot cystine 60 (11) The oxidation of cysteine 62 (111) The o~idation ot methionine 63 (iv) The oxidation of tyrosine 63 (v) The oxidation ot tryptophan 64 The Oxidation ot Soluble Proteins 65 (1) The oxidation ot insulin 65 (a) Oxidation by potassium permanganate 66 (b) Oxidation by chlorine 66 (11) The oxidation of lysozyme 68 (a) Oxidation by potassium permanganate 68 (b) Oxidation by chlorine 69 Conclusions 70 Page

CHAPTER III. A POLAROGRAPHIC INVESTIGATION OF THE OXIDATION OF CYSTINE, CYSTEINE AND TYROSINE 71 Introduction 71 Experimental Procedure 74 Results 74 (1) Oxidation by potassium permanganate 74 (11) Oxidation by chlorine 76 (111) Oxidation by potassium permanganate in the presence of sodium chloride 77 (iv) The polarography of partial oxidation products

of cystine 78

(v) A polarographic examination of the oxidation of cysteine by potassium permanganate 80 (vi) The spatial configuration of the disulphide bond in relation to its reactivity in proteins 81 (vii) Oxidation by sodium persulphate 86 (viii)Ox1dat1on by peracetic acid 86 Conclusions 86

CHAPTER IV. THE USE OF ULTRAVIOLET SPECTROSCOPY TO STUDY THE EFFECTS OF OXIDIZING AGENTS ON AMINO ACIDS AND PROTEINS 87 Introduction 87 Difference Spectrophometry 90 Experimental Procedure 92 Page

The Oxidation of The Amino Acids 94 1. The oxidation of tryptophan 94 (i) Oxidation by potassium permanganate 96 (11) Oxidation by chlorine 97 (iii) Oxidation by sodium persulphate 97 (iv) Oxidation by peracetic acid 99 (v) Oxidation by permonosulphuric acid 100 (vi) Interpretation of spectral changes 100 2. The oxidation of tyrosine 103 (1) Oxidation by potassium permanganate 104 (11) Oxidation by chlorine 106 (111) Oxidation by sodium persulphate 106 (iv) Oxidation by peracetic and permonosulphuric acids 106 3. The oxidation of cystine 107 The Oxidation of Soluble Proteins 108 1. The oxidation of insulin 110 (1) Oxidation by potassium permanganate 110 (ii) Oxidation by chlorine 111 (iii) Oxidation by sodium persulphate 112 (iv) Oxidation by peracetic acid 113 (v) Oxidation by permonosulphuric acid 114 2. The oxidation of casein 114 (1) Oxidation by potassium permanganate 114 (11) Oxidation by chlorine 116 Page

(111) Oxidation by sodium persulphate 118 (iv) Oxidation by peracetic acid 119 (v) Oxidation by permonoaulphuric acid 119 3. The oxidation of lyeozyme 120 (1) Oxidation by potassium permanganate 120 (11) Oxidation by chlorine 121 (111) Oxidation by sodium persulphate 121 (iv) Oxidation by peracetic acid 122 (v) Oxidation by permonosulphuric acid 123 (vi) General conclusions 123 The Oxidation of Fibrous Proteins 124 1. The oxidation of silk fibroin 125 (1) Oxidation by potassium permanganate 126 (11) Oxidation by chlorine 126 2. The oxidation of wool keratin 127

CHAPTER V. THE FORMATION BY OXIDIZING AGENTS OF FREE RADICALS IN WOOL AND SILK 129 Introduction 129 Detection of Free Radicals 130 Electron Spin Resonance Spectroscopy 131 The Formation of Free Radicals in Proteins 134 Experimental Procedure 138 Results 141 (a) Modification of the disulphide bond in wool 142 (b) Modification of the terminal and free amino Page

groups in wool 146 (c) Modification of the combined tyrosine 147 (d) Modification of the free carboxyl groups in wool 149 Chemical Analyses of Irradiated and Oxidized Wool 150 Conclusions 154

SUMMARY 157

APPENDIX 170

REFERENCES 173 ABSTRACT

The chemical changes produced by oxidation of wool keratin and related proteins have been investigated. Pre­ liminary investigations indicated that only five of the constituent amino acids of proteins are susceptible to oxidation under mild conditions and these are cystine, cystei~e, methionine, tyrosine and trYPtophan. From the fact that potassium permanganate and chlorine differ mukedly in their rates of attack of cystine and tyrosine, it was postulated that preferential oxidation of either of these amino acids may occur and this was subsequently shown empirically. The oxidation of cystine by potassium perman­ ganate has been found to proceed through the formation of a manganese (III) complex which is extremely unstable in the presence of halide ions. The possibility that a relationship exists between the steric configuration of the disulphide bond and the reactivity of protein-bound cystine is discussed. The oxidation of soluble proteins indicates that prefer­ ential oxidation of combined amino acids occurs. Thus, for insulin it was shown that potassium permanganate and sodium persulphate attack tyrosyl residues preferentially, whereas with chlorine preferential oxidation of cystine appears to occur. For proteins containing tryptophan in addition to cystine and tyrosine (for example, casein and lysozyme) it appears that potassium permanganate, chlorine and sodium persulphate initially attack only the combined tryptophan while with peracid oxidations it appears that the cystyl residues are oxidized simultaneously with tryptophyl. The formation by partial oxidation of free radicals in wool and silk has also been investigated. This study has indicated that the free radical locations in wool are associated with a number of amino acid residues, in particular cystine and tyrosine and possibly tryptophan. Furthermore, from the similarities in the structures of the ESR spectra of oxidized and 'lf-ray irradiated wool it was concluded that high-energy irradiation of proteins produces similar effects to oxidation. - l -

Chapter l: Ill'l'BOJJJCTIOlf

The effect ot oxidizing agents on wool is of interest tor two reasons. Pirstly, oxidizing agents are often used to bleach discolourations occurring in wool and secondly, certain oxidizi~ agents can be employed to render wool unshrinkable. Discolourations occurring in raw wool are usually caused by foreign matter and these are normally removed during scouring. A:n7 subsequent discolouration of wool, however, is usually a consequence ot some chemical modification of the wool keratin and its removal necessitates a bleaching treatment. 'l'h1s aspect of the work was not considered in the present inTeati­ gation, since the chemistry of bleaching requires a prior knowledge of the chemical nature of the pigments responsible and at present this knowledge is lacking. The main interest in this investigation, therefore, concerns the reactions occurring during oxidation since some oxidizing agents are known to produce a shrinkprooting finish whilst others are quite without effect. It it is assumed that oxidative ahrinlcprooting results from some chemical modification of the fibre at the molecular level then a study of the reactions occurring between wool and various oxidizing agents may shed some light on the mechanisms involved in the oxidative ahrinkprooting of wool. !he Chemical Rature of Oxidation

A number of reviews on the nature of oxidation have appeared11218• which have given precise explanations of the mechanisms involved in the case of inorganic compounds. However_, no corresponding description of organic oxidation has been published. Oxidation is a term used to classify a large number of diverse chemical reactions and quite often the reasons for the classification are somewhat obscure. In general, oxidation may be classified as a complete or partial with­ drawal of an electron (or electrons) from a chemical species, whilst reduction is the gain of electrons. Por inorganic compounds this definition is fairly accurate since, for example, the oxidation of iron to iron (II) and thence to iron (III) may be represented as follows: Pe '= ---+ :re+++ The electronic configurations of these species may be represented as follows: Pe ls2 2s2 2p6 3s2 3p6 3d6 4a2 Pe++ la2 2s2 2p6 3s2 3p6 346 Jle+++ ls2 2a2 2p6 3s2 3p6 346 The valence electrons (underlined) are associated only with the iron atom; in other words, they are not involved in - 3 - bonding with any other atoms. The removal ot these electrons, theretore, depends mainly on overcoming the attractive torce ot the nucleus and thia is uaually achieved by the introduction to the system ot a chemical species with a very high electron• attinity. With organic compounds a ditterent state of attairs exist,, since most of the valence electrons are shared with other atoms. Consider the case ot a typical alkane, tor example, ethane, in which both carbon atoms have all their valence electrons shared with other atoms. The electronic configurations ot the carbon atom may be represented as tollows, 28 2p C (ground state) ls2 282 2p2 [IT] ,~ IJ I C (reactive state) ls2 2sl 2p3 [O IJ IJ IJ C (as in ethane) la2 2a2 2p6 lul IJ dJ ~'IJ ~ I Atoms directly bonded: C B H H The dotted arrows represent electrons contributed by the other atoms. The removal ot a valence electron trom ethane clearly cannot occur in an analogous manner to that of iron. nectron abstraction in this case would mean the disruption ot a covalent bond and since carbon-hydrogen bonds do not favour ion tormation, bond rupture would occur by a homolytic path, where the dot signifies an unpaired electron. Thus, the electron is removed together with a proton so that the net loss is one hydrogen atom, resulting in dehydrogenation, which can be considered as the organic equivalent of inorganic oxidation. In this case homolysis results in the formation of free.radicals, which are chemical species modified so that they possess unpaired electrons. In the case of unsaturated organic compounds, such as ethylene, only three of the four valence electrons of the carbon atom are used in covalent bonding with other atoms. The electronic configuration of the carbon atom may be represented as, 2s 2p px py pz

C (as in ethylene) ls2 2s2 2P6 w ~--/J HJ I Atoms directly bonded: C H H The removal of any of the bonding electrons (sp2 hybridization) involves the disruption of a covalent bond and this is very difficult to achieve. It is somewhat easier to envisage the removal of a non-bonding pz electron for, here, only the attractive force of the carbon nucleus and the energy associated with the 1T- bond must be overcome. In this way, the system is similar to the inorganic analogue, but complete removal of this non-bonding electron does not usually occur, probably because of the energies involved in forming a carbon cation. Heterolytic fission, however, may take place in sane inataDCea. In the case of carbon bonded to an atom X ot somewhat large electronegativity, the carbon-X bond may with suitable stimulus, such as the addition of an electrophilic agent, undergo heterolysis,

In the case of inorganic atoms such as sulphur, oxygen, nitrogen and chlorine incorporated into organic molecules, the electron-affinity of these atoms must be considered in determining the ease of oxidation. Bulphur in its bivalent form (c-s-c) has an electronic coDf'iguration which may be represented as follows, 3s 3p S (bivalent) ls2 2s2 2p6 3s2 3p6 [] IJ r!J dJ rl Atoms directly bonded: C C In its valency shell sulphur has two bonding electron pairs and two lone pairs of electrons and evidently the removal of one of the lone pairs should be easier to achieve than removing one ot the bonding pairs. Bivalent oxygen is electronically similar to sulphur, 28 2p 0 (bivalent) la2 2s2 2p5 [] IJ rP tlJ j·I Atoms directly bonded: C C However, in this case, as a result of the smaller size of the oxygen atom, the lone pairs of electrons will be more firmly bound than those on the sulphur atom and therefore, they will be more difficult to remove. The direct removal of electrons tran an organic compound to tom a carbon cation can be produced by artificial means, tor example, by irradiating the compound with high-energy 'lS or X-ray radiation:

Thus, high energy irradiation of organic compounds may be considered to be a very pure form of oxidation since only the compound to be oxidi1ed is present in the system. A more detailed description of the interaction of high-energy radiation with proteins and its similarity to ox1dation processes will be presented later. So tar the discussion has been confined to complete electron withdrawal, however, oxidation may also proceed by a partial electron withdrawal. Consider the reaction of the halogen atoms with the alkanes, as represented by the equation, ultraviolet CJl.4 + 012 light > CB3-Cl + HCl

The chlorine has removed a hydrogen atom from the methane to form methyl chloride. This may be considered as a partial oxidation since the highly electronegative chlorine atom has drawn the bonding pair of electrons to a position much f'urther - 7 - removed trc:m the carbon atom than the other three bonding pairs, r r CH3 ----> Cl and as a result the molecule is dipolar. Hydrolysis ot methyl ~hlor1de produces methanol, which is an oxidative product of methane, so that methyl chloride and methanol may be considered to be in the same oxidation state.

The Bature of Oxidizing yents Oxidizing agents may be classified as species possessing extremely high electron affinity and the reason for this can be attributed to the tendency of the species to reach a more stable electronic configuration or, in other words, to lower its chemical potential. Consider the case of manganese, its electronic configuration may be represented as, 3d 4s 11n ls2 2s2 2p6 3s2 3p6 3d5 4s2 IJ IJ IJ IJ IJ I [] The loss of the two 4s electrons results in the formation of the manganous ion, 3d 2 Mn + ls2 2s2 2p6 3s2 3p6 3d6 WIJ IJ IJ IJ This configuration corresponds to a transitional metal ion electronic structure4 and since the five 3d orbitals are all half-filled this structure is relatively stable electronically. It is possible to remove the five 3d electrons to form a +7 oxidation state, such as that occurring in the cc:mplex - 8 - permanganate anion (lln 04-), 11n 7+ 1a2 2a2 2p6 3a2 3p6 3do 4a0 This configuration, however, ia not as stable as the manganous state (probably associated with the very high unbalanced nuclear charge) and the 11n 7+, therefore, will try to return to the electronic state ot 11n 2+. To achieve this, the 11n 7+ mu.at obtain five electrons and this is accomplished by withdrawal ot electrons trcm an atom or atoms ot a molecule having very low electron-affinity. In the case ot the halogens, several electronic structures are possible, the more stable ones being the shared electron-pair configuration, which may be attained as follows, 3s 3p px PY pz 2 2 2p6 2 Cl la 2a 3a 3p5 [] IJ rµ ~, rl ~ ~e!;~J: are Cl la2 2s2 2p6 3s2 3p5 [] IJ tlJ ~IJ I ~~:e~1~0 torm Bach ot the unpaired electrons is shared by both chlorine atoms and this simulates the inert gas electronic configuration. ot argon. However, the most stable electronic configuration that chlorine can attain is that ot the chloride ion (01-),

tor, in this case, each chlorine atom has achieved the inert gas electronic configuration completely and separately, Cl - 1,JZ 2s2 2p6 3a2 3p6 _ A Therefore, given the choice, chlorine will tend to asaume the - 9 - electronic cont'iguration of the chloride ion by abstracting an electron frcm acme other speciea. Certain oxidizing agents such as the persulphate anion and the ferrous ion-hydrogen peroxide system (Fenton's reagent) give rise to free radical species, such as so4•• and O:S- respectively in solution. As a result of the unpaired electron, these species are extremely reactive and in order to pair-off this odd electron they tend to remove electrons or hydrogen atoms from other chemical species. Another class of oxidizing agents is that of the pero.xy canpounds and their reactivity depends on a number of factors. Essentially, however, the chemical reactivities of both hydrogen peroxide and the per-acids depend on the structural fact that the O - 0 covalence is a weak bond, which can be fairly easily severed to give either a pair of reactive hydroxyl radicals (OH"') or, alternatively, a stable hydroxide anion (:OH-) plus a reactive hydroxyl cation (OH+). The reactivities of these species are again associated with an unstable incomplete electronic configuration. From the above discussion two different oxidation processes may be formulated: 1. Intermolecular oxidation. 2. Intramolecular oxidation. 'l'he former involves the complete removal of electrons fr0111 a chemical species by some other species attempting to achieve - 10 - a more stable electronic configuration. The latter results fran a partial withdrawal of electrons between atoms within the one molecule or species due to a difference in the electron affinities ot the respective atms.

The Chemical Nature of Proteins Chemically, proteins are elaborate polypeptides formed by the linear condensation ot C.X-amino acids as follows:

R2 I + :NH2.CH.COOH > R2 I ..... NH. CH.I CO. NH. CH. CO ..... + ..... H20 ..... The nature of the side chains R which are attached to the Cl-carbon atoms of the amino acids incorporated in the polypeptide chains is held to be responsible for the differences in physical and chemical properties between various proteins. Some twenty amino acids have been isolated and identified from proteins after hydrolysis of the peptide linkages with acid or alkali solutions. It is convenient to subdivide these amino acids into seven classes according to the nature ot the respective side chains. These are described below and the structure of the side chains are shown in Table 1.1. Also included is the amino acid composition of Merino 64's - 11 -

TABLE 1.1 nature of Amino Chemical Structure Amino Acid Content Side Chain Acid ot side chain R Wt./lOOg. dry wool S111:monds Cortield & Robson P.ARAFFINIC glycine H- 5. 2 5. 6 alanine CH3- 3. 7 4.4 valine (CH3)2 CH- 5. 0 5. 8 leucine (CH3)2 CH.CH2- 7. 6 9. 1 isoleucine CH3.CH2.CH (CH3)- 3. 1 3. 8 BASIC lysine NH2• (CH2)4 - 2.a 3. 3 arginine NH=C(NH2 ).NH.(CH2)3- 10. 5 9. 9 ACIDIC aspartic HOOC.CH2- 6. 7 6.9 glutamic HOOC.CH2.CH2- 15. 0 14.8 AROMATIC phenylalanille OCH2- 3.4 4. 2 tyrosine ~H2- 6.4 5. 6 .ALCOHOLIC serine HO.CH2- 9. 0 10.a threonine CH3.CH(OH)- 6. 6 7. 3 DTERO- tryptophan ~CH2- 2. 1 1. 0 OYOLIC histidine H ~H2- o. 9 1. 2 N::!,,.~C"" NH proline QcooH H 7. 3 6. 9 H hydroxy- HYcooH proline ... SULPHUR cystine -CH~S-S-CH2- 11.3 10.5 CONTAINING cysteine HS.CH2- 0.4 methionine CH3.S.(CH2)2- o. 7 o. 6 - 12 - as determined by S1mnonds6 and by Cortield and Robson6• Claes 1. Amino acids containing parattinic side chains. The amino acids ot this group are glycine, alanine, Taline, leucine and isoleucine. Thie group constitutes approximately 24- ot the total residues in wool but they are unreactive and do not play an important role in the chemistry ot proteins. Clase 2. Amino acids containing basic side chains. 'l'hese are the dibasic mono-acidic acids, arg4n1ne and lysine, which yield the strongly basic guanidino and amino groups respectively. Class 3. .Amino acids yielding acidic side chains. 'l'hese are the monobasic diacidic acids, aspartic and glutamic acids, which yield side chains containing a terminal carboxyl group. Class 4. The arcmatic amino acids. !he amino acids ot this group are phenylalanine and tyrosine. '?he tormer is relatively stable due to the stability ot the benzene ring whilst the latter is very labile as a consequence ot the phenolic hydroxyl group in the ring. Class 6. '?he /3 -hydroxy amino acids. '?his group consists ot the amino acids serine and tbreonine which possess the reactive primary and secondary alcoholic groups respectively. Hydroxyproline may be included in this group since there is an alcoholic hydroxyl group on the number tour carbon atom. - 13 -

Class 6. The heterocyclic amino acids. This group consists ot the amino acids trJptophan and histidine and the 1m1no acid proline. ~e former two are very labile as they possess the reactive indole and 1m1dazole ring structures respectively. Class 7~ The sulphur containing amino acids. This group contains cystine, cysteine and methionine ot which cystine is by tar the most abundant and the most reactive. Cystine is found abundantly in all keratin fibres and plays an important role in determining their properties as compared with those of other proteins. The classification presented above is not rigid, histidine tor instance yields a basic reaction due to its imidazole group in its side chain and therefore, may also be classified as a basic amino acid. Tryptophan, although strictly a heterocyclic compound, is often classified as an aromatic amino acid, while proline and hydroxy-proline are not amino acids but imino acids since their o<.-amino group is incorporated into a ring structure. The intermolecular forces responsible for holding the polypeptide chains together have been postulated as follows: (1) Generalized attraction due to van der Waals forces. These do not play a very important role in some proteins because the relatively bulky side chains present, restrict close packing and tend to keep the polypeptide chains too far - 14 - apart tor these forces to be very effective. (11) Hydrogen bonding - this is said to take place between the lffl groups ot one chain and imnediately adjacent CO groups ot neighbouring chains. Bot all :RH and CO groups in proteins are 11nlced1n this way, since here also the bulky side chains may keep many ot the main polypeptide chains separated to such an extent that hydrogen bonding is seriously hindered. 1'1 th proteins in which the molecular chains are helical, e.g., keratin, hydrogen bonding between CO and ml groups are intra­ chain to stabilise the helix. (111) Salt linkages - this type ot linkage, first suggested by Spealanan and H1rst7' is formed between adjacent chains by electrostatic attraction between a charged carboxylic group in one chain and an adjacent charged amino group in a side chain of the other. Such electrostatic forces are completely disrupted by acids and alkalies as shown,

Alkali

{iv) The Disulphide Bond - this is the covalent bond linking two adjacent polypeptide chains owing to the amino acid cystine: - 16 -

coI coI ml lUI I I CB - CB2 - S - S - CB2 - CB Cystine residue I I co co lffl ml I I It is this linkage which characterizes all keratin tibres by imparting the properties ot water insolubility and mechanical strength. (v) Hydrophobic bonds. In addition to the intermolecular torces already mentioned, there is some evidence to indicate that additional torces operate in aqueous media8• These torces have been termed "non-polar" bonds or hydrophobic bonds and may be considered to be additional van der Waals torces, which exist only when the protein is dissolved or swollen in an aqueous media. These "non-polar" bonds arise trom the tendency ot the non-polar side chains ot the protein to aggregate micelle-like in the interior ot the protein molecule to withdraw trom the aqueous medium ot the solvent or the swelling agent. Proteins which consist wholly ot polypeptides are usually referred to as simple proteins and these may be further subdivided into globular and fibrous proteins. Globular proteins are somewhat rounded in shape but not necessarily spherical and they are particularly prone to denaturation. - 16 -

Pibrous proteins, on the other hand, are relatively insoluble and exhibit a highly organized internal structure which permits a more systematic packing of molecular chains and hence greater cohesion between them from polar and ionic bonds. Pibrous proteins may be divided into elastic or coiled proteins and inelastic or extended proteins. The first group includes the keratin of wool, feathers, horn etc., while the second group includes the tibroin ot silk and the collagen of skin. Elastic proteins when fully extended under mechanical load have similar characteristics to those ot inelastic proteins. They are presumed to have in the coiled state a helical structure stabilised with hydrogen bonding between adjacent turns of the helix. Proteins associated with non-proteinaceous material are termed conjugated proteins and these may be classified as follows: (a) Phosphoproteins, in which the protein molecule is bound to phosphoric acid (which may itself be part of the bigger organic complex) through an ester grouping. Casein is an example. (b) Glycoprotein, or mucoprotein, in which the protein molecule is bound to ca~bohydrate. (c) Hucleoproteins, which are conjugates ot protein and nucleic acid. (d) Chromoproteins, which are conjugates of protein with - 17 - coloured materials and are concerned with oxidation - reduction systems. (e) Metalloproteins, which also take part in oxidation - reduction systems. Haemoglobin is an example ot this and would also rank as a chromoprotein. (f) Lipoproteins, which are conjugates ot protein with fat­ like substances. Since proteins are comprised of such a large number of amino acids, complex analytical methods are required tor the estimation of each amino acid present. Early analytical procedures involved the isolation of a particular amino acid from the hydrolysate of the protein by precipitation and fractional recrystallization and its subsequent estimation by colorimetric or gravimetric techniques. The advent of paper chromatography provided a simple method tor separating the individual amino acids and it is used extensively tor qualitative work9,lO. Its adoption tor quantitative analyses, however, has not been as success:f'ul, since difficulties are experienced in the complete separation and elution of the spots obtained. Ion exchange chromatography employing traction collectors11 have effected complete separation of the amino acids but their subsequent colorimetric analysis is a long and tedious operation. This last deficiency has been overccae by the use of automatic traction collectors employing automatic colour development am estimation. Present research - 18 - on this problem includes the application of high voltage electrophoresis, gas chromatography and mass spectrometry.

One of the main problems which besets chemical methods of amino acid analyses of proteins is the prerequisite for hydrolysis to convert the polypeptide into its free individual amino acids. In addition to peptide bond breakdown the hydrolytic procedure can also cause some degradation of the amino acids, for example, tryptophan, serine, threonine etc., depending on the individual amino acid as well as the conditions of hydrolysis. This hydrolytic degradation of the amino acids can be partly accounted for by determining their losses during a simulated hydrolysis of standard solutions of mixtures of the amino acids. From this a correction factor can be obtained to allow for damage occurring to the amino acids during hydrolysis of the protein. Clearly, however, this is suitable only for chemically unmodified proteins, since with modified proteins there is no way of predicting the behaviour of all the modified amino acid residues towards hydrolysis. Thus, methods of analyses, not involving primary hydrolysis, are most desirable. To this end some progress has been made with the amperometric determinations of cystine or cysteine in intact proteinsl2. H-terrninal and a-terminal analyses of wool keratin have shown that wool is comprised of a large number of proteins. It has been demonstrated that the amino acids glycine, alanine, - 19 - serine, threonine, valine, glutamic acid and aspartic acid constitute the B-terminal groups13' 14• These measurements have yielded a figure of approximately 60,000 as the average mtlecular weight of the protein chains in wool. Recently it was shown that in wool a large number ot the N-terminal amino groups are masked by acetyl groups15, while additional evidence16 indicates that cystine should be included among the B-terminal groups and that the average molecular weight should be lowered to 36,000. The C-terminal amino acids appear to be glycine, alanine, serine and threonine17 but owing to certain deficiencies in the method of detection there are probably others. The fractionation ot wool into various components has been achieved by tirst disrupting the disulphide linkages, either by preferential oxidation or reduction, followed by extraction with alkali or with hydrogen bond breaking (disassociating) agents18, 19, 20, 21• Three components, known

as o(._, f.>- and o - keratoses, have been isolated from wool after conversion of the cystine groups to cysteic acid22• 15'- keratose has been found to have an extremely high sulphur content in comparison with the other two fractions and with intact woo1 23• It has recently been shown that cysteyl cysteic acid occurs in Ol- keratose but not in c5 - keratose24 and tran this it was concluded that the cystyl residues of low sulphur proteins occur in adjacent positions in the polypeptide chain - 20 - whereas those of the high sulphur proteins are more evenly spaced along the chain. Zahn and his coworkera25 have found that in O{- keratose the amino acids arginine and lysine are predominantly linked with serine and the short chain neutral amino acids. Associated with its chemical complexity, wool is found to possess an extremely complex morphological structure. Thus, fine wool fibres can be divided into two distinct regions, the cuticle and the cortex, of which the latter constitutes over 90,C of the total. These regions are composed of different types of cells differing in chemical reactivity and in addition it has been found that each region may again be subdivided. Thus, the cuticle consists of the epicuticle, the exocuticle and the endocuticle, each differing in chemical and enzymatic reactivity. The epicuticle is a thin membrane covering the cuticular cells and it has been shown to be resistant to chemical degradation26 and to the penetration of dyes27.

The main body of the wool fibre is the cortex and with fine wools possessing bilateral as1J1111etry the cortex has been shown to consist of a reactive, less dense ortho-cortex and an unreactive, hard para-cortex. It has been found that the ortho and para-cortical cells differ in chemical compoeition28, in particular the para-cortical cells are richer in cystine than the ortho-cortical cells. The cortical cells can be - 21 - further subdivided into microtibrila and matrix which also differ in structure and chemical canposition, for example, the matrix has been found to have a high sulphur content. Fraser29 has suggested, from X-ray diffraction and electron microscopic evidence, that the microfibril is canposed ot eleven prototibrils, each comprising of three CX.- helices, arranged so that in cross-section there are two protofibrils surrounded by an outer circumference of nine more protofibrils. Differences in microfibril arrangement between ortho- and para­ cortical cells have been suggested as being responsible for the differences in their sulphur contents, the para-cortical cells possessing more matrix between the microfibrils30 thus accounting for their higher cystine contentlS. It is also thought that the high sulphur proteins (1S- keratose) fractionated from wool arise from the matrix whilst the low sulphur proteins (ci- and f3 - keratoses) arise tran the microfibrils. This picture to some extent correlates the molecular and morphological structures ot wool, but it does not provide explanations tor all the observed phenomena so that at present it cannot be considered as complete. Since methods have not yet been developed for the isolation of the homogeneous component proteins of wool, investigations into the chemical reactivity and degradation of wool must treat the fibre as a whole. - 22 -

The Oxidation of Amino Acids and Proteins From theoretical considerations a large number of the constituent amino acids of proteins would be expected to have aide chains susceptible to oxidation because of localized high electron density coupled with relatively low electron affinity. Por discussion these amino acids can be classified into a number of groups: Group 1. Side chains containing tertiary carbon atoms. The amino acids leucine and isoleucine have tertiary carbon atoms which may be oxidized to the corresponding tertiary alcohols31 •

(o] - H )

Group 2. Side chains containing primary or secondary alcoholic hydroxyl groups. Th.ere are two amino acid residues in proteins possessing such groups, namely serine and threonine. It is well established that primary alcohols are easily oxidized to aldehydes and subsequently to carboxylic acids whilst secondary alcohols are oxidized to ketones32•

(O] R.CH2.0H ) R.CHO [oJ > R.COOH R (O] R and 'cH - OH ) .,.,.'c = 0 R"" R Group 3. Side chains with ring structures. The amino acids of this group are histidine, tyrosine, - 23 - tryptophan and the imino acids proline and hydroxyproline. Phenylalanine is not included since fairly severe treatments are necessary tor its oxidation. The ring structures of the remaining amino acids would be expected to undergo substitution and/or fission during oxidation. Group 4. Side chains containing sulphur atoms. This group comprises the amino acids cystine, methionine and cysteine all of which should be susceptible to oxidation since they contain bivalent sulphur with lone-paira ot electrons capable of being partially withdrawn by an oxidant33: 0 0 [O] > -~- -s.·.- ~ -~-•• I 0

The free E: -amino group and the guanidino group of the amino acids lysine and arginine respectively could undergo oxidation, possibly to amine oxides, but salt formation ot these groups in acidic media usually providessome degree of protection against oxidation34• It is evident from the above discussion that at least twelve of the twenty constituent amino acids of proteins should have oxidizable side chains, yet chemical analyses of oxidized keratin indicate that, in general, only the amino acids cystine, tyrosine, tryptophan and methionine are prone to oxidation, although modification of lys1ne35, histid1ne36, serine37 and proline36 has been reported. The stability ot the remaining amino acid residues is difficult to reconcile with the theory. This is particularly so in the case of serine and threonine when the relative ease of oxidation of primary and secondary alcohols is considered. The stability to oxidation of leucine and isoleucine, on the other hand, is probably associated with the rather severe treatments necessary to oxidize tertiary carbon atome38• The following discussion is not intended to be a detailed survey of the oxidation of proteins but is a review of the present state of knowledge concerning the oxidation of the various reactive groups present in wool keratin. (1) The oxidation of cystine. A survey of the literature on the oxidation of wool keratin indicates that, in the majority of cases, this has been investigated on the basis of combined cystine reactivity - a possible consequence of the Speakman, Nilssen and Elliott hypothesis39 regarding a relationship between oxidative shrink-proofing of wool and cystine modification. The oxidation of the disulphide bonds of the cystyl residues in keratin has been investigated in detail in recent years and the main oxidizing agents employed in these studies have been peracetic and performic acids. These reagents do not, in general, react with all the amino acids but have a preferential reaction with cystine, tryptophan and methionine40• Complete oxidation of free cystine by these reagents give - 26 - quantitative yields of cysteic acid40• HOOC Cy.S.S.Cy (Cy = >H-CH2-) lffl2 However, with incomplete oxidation a variety of oxidation states, intermediate between the disulphide and the sulphonic acid, is possible and these are shown in Table 1.2.

TABLE 1.2 Possible Oxidation Products of Disulphides Oxidation level Products (R.s.s.R. = o) Intact s-s Bond s-s Bond Fission -1 R.SH

0 R.S.S.R l R.S.SO.R (I) R.SOH 2 R.SO.SO.R; RS.8O2.R (II) 3 R.SO.8O2.R 4 R.SO2.8O2.R (III)

5 The only intermediate oxidation products of cystine which have been isolated are the a-monoxide (I)41 and the S.S-dioxide (II )42,43. These compounds are generally unstable in aqueous solutions. In neutral and alkaline conditions both the cystine monoxide41 and dioxide43 are quantitatively hydrolysed to cystine and alanine-3-sulphinic acid,

3 Cy. SO. S. Cy + H20 ~ 2 Cy. S02H + 2 Cy. S.S. Cy 3 Cy. s02. s. Cy + 2H~~4 Cy. so~ + Cy. s. s. Cy - 26 -

Hydrolysis to the same two products occurs in acidic media but traces of cysteic acid are also formed. Disproportionation of the cystine monoxide to cystine and to cystine dioxide is also found to occur and this is particularly rapid in the presence of chloride ione44, 2 Cy.SO.S.Cy-->Cy.S.S.Cy + Cy. 802- S. Oy Oxidation of the cystine monoxide with one equivalent of peracetic acid in water or dilute sulphuric acid at o0 c, gives cysteic acid, unreacted monoxide and only traces of cystine dioxide and alanine-3-sulphinic acid, although with aqueous hydrochloric acid cystine dioxide appears to be the major product44• 'l'he main product of keratin cystine oxidation appears to be cyste1c acid, the presence of which has been demonstrated in intact woo145 and in hydrolysates of wool oxidized by hydrogen peroxide46, peracetic acid47, 4s,49, potassium permanganate47,48, chlorine46, 48,60, bromine46 , potassium persulphate36 and by many other ox1dants51,62,63,54. Alexander, Fox and Hudson48 , in their studies of the oxidation of wool with peracetic acid, were able to account for all the cystine in terms of cysteic acid, but were unable to demonstrate the presence of sulphonic acid groups in the intact fibre. These workers suggested, therefore, that the oxidation product in the intact fibre was a cyclic eulphocarboxyimide which on acid hydrolysis yields cysteic acid quantitatively, - 27 -

The presence of sulphonic acid groups in oxidized keratin has since been unequivocally demonetrated45, but the lack of ion-exchange properties corresponding to these groups indicates that in keratin they are ionized and internally compensated by ionized basic groups55. The formation of intermediate disulphoxides in partially­ oxidized keratin has been suggested56 and confirmed by paper­ chromatography57, but infra-red spectrscopy has revealed that these "disulphoxides" are in fact thiolsulphonates58, 69, 60• Under severe oxidative treatments a decrease in the total sulphur content of the fibre often occura48, indicating carbon-sulphur bond fission. Early investigations of the oxidation of wool were concerned with estimating the amount of cystine modification and this was usually obtained by quantitative analyses of - 28 - cystine before and after treatment. These analytical methods required an initial hydrolysis of the protein to its constit­ uent amino acids and provided this hydrolysis was carefully controlled to ensure minimal cystine losses, a fair estimate of the cystine content was obtained. With the advent of a non-hydrolyt1c procedure for estimating the disulphide content of intact proteins12, it appeared that, in most oxidations of wool, a proportion of partially oxidized cystyl residues was present in the intact fibres49• This is evident fran the results shown in Table 1.3, where a comparison of the hydrolytic and non-hydrolytic procedures for disulphide analyses of oxidized wool has been made.

TABLE 1.361 Dilsuphide and Thiol Contents (µ. moles/g. dry wool) Wool Intact Hydrolysed Condition of treatment A L _Q_ Untreated 491 456 444 0.0614 peracetic acid for 1 h. 105 318 314 0.13M peracetic acid for l h. 47 192 189 0.2611 peracetic acid for 2 h. 22 82 77 0.2511 peracetic acid for 24 h. 11 37 36

A: by the method of Leach62 B: by the method of Shinohara63

C: by the method of Stricks, Kolthoff and Tanaka64 - 29 -

The results in Table 1.3 can be explained by asswning that partially oxidized cystyl residues are present in oxidized intact wool. The increase in disulphide content after acid hydrolysis then arises from disproportionation of these partially-oxidized residues and therefore, the discrepancy between the hydrolytic and non-hydrolytic methods may give some measure of the amount of partially oxidized residues present in woo166. For wool oxidized with peracetic acid these partially­ oxidized cystyl residues appear to be S-monoxycystyl or s.S-dioxycystyl, corresponding to cystine monoxide and dioxide respectively, since oxidized wool has been found to react with a number of reagents in a similar manner to these tree amino acid analogues44• In the light of this evidence and fran the tact that peracid oxidation of a number of proteins, including wool, always results in sane degree of incomplete oxidation, the validity of results of early studies of cystine modification in oxidized wools must be treated with some reservation. In this connection a derivative of cystine, alanine-3-sulphinic acid, was found to suffer no degradation when subjected alone to normal hydrolytic conditions but hydrolysis in conjunction with the amino acids comprising wool lead to its complete destruction, thereby indicating that alanine-3-sulphinic acid combines with one or more or the naturally occurring amino acids of woo166. - 30 -

A drawback of the non-hydrolytic amperanetric method is that both cystine monoxide and peracid oxidized wool react with methyl mercuric iodide in the same way as thiols,

R.SH + Me.Hg.I ----4 R.S.Hg.Me + HI, and R.so.s.R +Me.Hg.I+ H20----+R.S.Hg.Me + R.S02H + HI, so that as yet it is not possible to differentiate between S­ monoxycystyl residues and true thiols in oxidized woo167• Another interesting facet of the oxidation of wool is the observation that some oxidants (hydrogen peroxide68 , peracetic acid48 and chlorine48 are capable of oxidizing all the cystine, whereas others (acid and alkaline potassium permanganate47, alkaline hypochlorite47) can attack only 25~ of the total cystine. Thie is difficult to reconcile with reactions on model compounds, where it is found that disulphides are oxidized very readily in the cold68. It has been suggested that oxidizing agents in the form of anions can only react with a 25% fraction of cystine, whereas unionized molecules can remove all the cystine68 , but this suggestion has no theoretical basis. Explanations of this behaviour based on morphological differences, electrical repulsion and crystall­ inity of the fibre have been rejected47. Side chain environment does not appear to be responsible since peptides of varying amino acid composition are readily oxidized by chlorine and by hypochlorite ions69. A possible explanation, not previously proposed, may lie in the steric configurations - 31 - of the disulphide bond and this will be discussed in more detail later. (11) The oxidation of tyrosine. Under degradative conditions, oxidation of tree tyrosine occurs by selective degradation of the alkyl side chains to form aromati~ aldehydes70 and carboxylic ac1ds71 , formic and aapartic acids. With milder conditions the side chain can remain intact and oxidation of the phenolic ring to 3,4- dihydroxyphenylalanine occurs when hydroxylating agente such as hydrogen peroxide72 and Fenton's reagent73 are used. Cyclization of the alkyl side chains to form indole derivatives has also been reported during oxidations of free tyrosine with potassium ferr1cyan1de74 and eerie ammonium sulphate76• The reaction of tyrosine with the halogens results in the formation of 3,5-disubstituted halogen derivatives76. 'l'he oxidation of combined tyrosine in wool probably proceeds initially by attack on the phenolic ring since, here, the alkyl side chains are firmly incorporated into the protein main chains. Thus, combined tyrosine in wool reacts with iodine to form 3,5-d1iodotyros1ne77• D1chlorotyros1ne has been isolated from proteins oxidized by peracetic and performic acids78,79 in the presence of hydrochloric acid or by the subsequent treatment of the oxidized protein with hot hydrochloric acid. Thompson78 believes that the dichloro- tyrosine arises fran residual hydrogen peroxide in the system - 32 - oxidizing the chloride present to chlorine which then substitutes the phenolic ring. This is supported by the tact that no chlorinated derivatives of tyrosine could be detected in proteins oxidized by pertormic acids in the absence of hydrochloric acid and hydrolysed by sulphuric acid. Schirl, and Meybeck80 have made an extensive study of the reaction of wool with chlorine dioxide and chlorite in acid solutions. These workers suggested that the brown colouration produced was caused by oxidation of tyrosyl residues since free tyrosine yielded the same colour and they were able to isolate from oxidized free and combined tyrosine the melanin precursor, 5,6-dihydroxyindole-2-carboxylic acid. Earland, Stell and Wiseman81 consider that melanin-type cross-links are produced in proteins by oxidation of tyrosyl residues with potassium nitrosodisulphonate. These workers subjected oxidized proteins to alkaline hydrolysis and extracted into an organic phase a brown coloured residue which they subjected to ultraviolet absorption analyses. The formation of an indole from c0D1bined tyrosine is difficult to envisage since the alkyl side chains of combined tyrosine are not free to cyclize. It is probable, therefore, that the "melanin-like" compounds reported arose by residual oxidizing agent in the system oxidizing free tyrosine produced during alkaline hydrolysis of the proteins. Quinone formation arising from the oxidation of combined - 33 - tyrosine in wool has also been suggested. Das and Speakman52 consider that an orthoquinone is formed in wool oxidized by chlorine dioxide since tyrosine was found to be extensively modified by this treatment. AlexaDder, Carter and Hu.dson82 auggested that a para-quinone 1s produced by acid permanganate oxidation as the wool assumes a yellow colour which behaves as an acid-base indicator in an analogous manner to other quinoid structures. However, isolation of quinones from oxidized wool has not been achieved as yet. The discoloura- tion of oxidized wool has also been attributed to modified tyrosyl residues83 but this conclusion must be viewed with some reservation since tryptophan can also give rise to coloured oxidized derivatives84• Apart from the above studies, which are concerned with the possible fate of combined tyrosine during oxidation, it has been found that oxidation of proteins is often accompanied by the loss of tyrosine. Thus potassium dichromate85, potassium permanganate86, hydrogen peroxide35 and nitric acid87 all lower the tyrosine content of proteins. The combined tyrosine of wool is not equally reactive and in this way resembles the combined cystine. Thus some reagents, such as sodium hypochlorite and potassium permanganate, can react with 30% of that present in wool whereas others, such as chlorine and chlorine peroxide, can eventually remove all the tyrosine. Alkaline permanganate solutions can react with - 34 -

all the tyrosine and in this respect the tyrosine behaves differently from the cystine in wool, which is very resistant to attack by alkaline permanganate86. There is also some disagreement concerning the loss of tyrosine in chlorinated wool. Alexander and Gough86, employing th~ Lugg's procedure88 , have found that the tyrosine content of wool is extensively lowered by chlorination, whereas McPhee60 was unable to detect any such loss by employing the spectrophotometric method of Goodwin and Morton89 as modified by Simm.onds6• in conjunction with the colorimetric procedure using diazotized sulphanilic acid90• (111) 'l'he oxidation of tryptophan. 'l'he indole nucleus of free tryptophan is extremely susceptible to oxidation which usually proceeds by the disruption of the 2:3 carbon-carbon bond of the pyrrole ring and/or by substitution in the aromatic ring. In some cases weak oxidizing agents may oxidize tryptophan without disrupt­ ing the indole r1ng91 but the more powerful oxidizing agents, in general, disrupt the indole ring producing a variety of products such as N-formylkynuren1ne92, kynurenine93, anthranilic acid93 and various phenolic derivatives of these compounds. It is well substantiated that combined tryptophan in proteins is susceptible to oxidative modification, but apart from simple polypeptides94 the fate of the tryptophan in - 36 - oxidized proteins is unknown. The degradation of combined tryptophan in wool oxidized by hydrogen peroXide has been investigated but the results are conflicting. Mazingue, Decroix and van 0Verbeke95 and Miro96 have found that a large reduction of the tryptophan content occurs whereas Graham and Statham97 report only a very alight reduction in tryptophan content. The latter workers have suggested that the values obtained by Mazingue et a195 and by Miro95 could possibly arise from the presence of residual peroxide in wool which, on acid hydrolysis with hydrochloric acid, oxidized the chloride to chlorine which subsequently oxidized tryptophan. Miro99 has revised his work by employing sodium sulphite to remove any residual hydrogen peroxide from the wool before hydrolysis and he has shown that the lose in tryptophan is unaltered by the reduction treatment, in agreement with the results of Ziegler98• McPhee has briefly investigated the modifications occurring to wool treated with chlorine50 and with neutral potassium permanganate in saturated sodium chloride solutiona100 but he was unable to detect any loss of tryptophan. These findings, however, have not been supported by the work of Graham and Statham97 and the results of Andrews, Inglis, Rothery and Williams37 are inconclusive in this connection. Although no detailed investigation on the role of tryptophan during oxidation of wool has been attempted, it is known that - 36 - tryptophan is lost when wool is treated with potaasiwn dichromateB5, alcoholic iodine97, permonosulphuric acid97• potassium persulphate97 and peracetic ac1d97• However, despite the importance of tryptophan in the oxidation of wool it has sometimes been neglected completely36• (iv) The oxidation of the other amino acid residues. No detailed investigation has been carried out on the reactivity of the other amino acid residues in wool. Routine amino acid analyses of wool, treated with various oxidi~ing agents, have indicated that degradation of methionine101, aerine37 and proline36 occurs but the extent of degradation appears to be very dependent on the particular oxidizing agent employed. Claims that valine37, arginine37, glutamic acid37 and histidine36 are modified during oxidation, must be treated with some reservation due to the high experimental error inherent in the analytical method employed37• (v) Conclusions. Prom the preceding discourse it is evident that the oxidation of wool keratin is an extremely complex process and, despite the large number of investigations, some controversy still exists regarding the oxidizability of various amino acid residues. Thus, no general conclusions are possible but it would appear that the chemical modifications, occurring to wool during oxidation, are largely dependent on the particular oxidizing agent and the conditions under which it is employed. - 37 -

The purpose of this investigation was to detel'Dline the amino acid residues of wool most susceptible to oxidation and to investigate the possibility that the oxidation of wool may occur in a munber of preferential stages. The oxidations, in general, were carried out in acidic media to avoid complica­ tions arising from the conversion of cystine to lanthionine under alkaline conditions. It is clear that the majority of the previous work on the oxidation of wool has been concerned with the loss or gain of the constituent amino acids after oxidative treatments. The inadequacies of the analytical procedures employed in these investigations are generally associated with subsequent modification of the oxidized derivatives during hydrolysis of the protein to its constituent amino acids. These have already been discussed and will not be treated further. To overcome this problem of hydrolytic modification of oxidized derivatives of wool many investigators have studied the oxidation of model compounds and in particular the free amino acids. The procedure usually adopted involves the isolation and characterization of the products of the oxidation of a particular amino acid and subsequently, the chemical properties of oxidized wool, treated in an·analogous manner, are compared with the amino acid derivatives - a similarity in reactivity is usually taken as a criterion for the presence of the derivatives in the intact fibre. This approach eliminates - 38 - the possibility ot disproportionation during hydrolysis and has been successfully employed to demonstrate the presence of cystyl monoxide groups in intact wool partially oxidized with peracetic acid44• The possibility that several products may be formed from the oxidation ot one amino acid further complicates the issue. To overcome the inadequacies associated with the existing chemical procedures for analyzing oxidized wool, it was decided to investigate the oxidation of wool keratin indirectly, by studying the effect of oxidizing agents on model compounds, in particular the tree amino acids and a few soluble proteins. The oxidation of these model compounds may be studied by the application of such physico-chemical techniques as potentiometry, polarography and ultraviolet difference and absorption spectroscopy. Although a very large number of oxidizing agents are available for investigation, it was found necessary to restrict this study to the following five, potassiwn permanganate, chlorine (hypochlorous acid), permonosulphuric acid, peracetic acid and sodium persulphate. This list includes representa- tives of various types of oxidants as well as some important cormnercial shrinkproofing agents. - 39 -

Chapter II: THE OXIDATION OF AMINO ACIDS AND PROTEINS AS STUDIED BY POTBNTIOMETRY

Organic oxidation systems are generally irreversible although exceptions such as various quinone systems1O2 and oxidation-reduction indicators are known. These reversible systems appear to involve transfer of protons to or fran oxygen, sulphur and nitrogen atoms and this transfer is usually associated with facile addition or removal of electrone1O3• The covalent character of organic canpounds is responsible for the irreversibility of most organic oxidations and this in turn prevents the application of classical thermo-dynamics to the study of such systems. However, this deficiency does not exclude the employment of potentiometric techniques, the necessary requirement here is that the oxidizing agent is capable of oxidizing the compound in question and that the reduced and unmodified forms of the oxidant are distinguishable by the extent of their interaction with an inert detecting electrode.

The Theoretical Principles of the Method Potentiometric titrations in general employ a cell with an inert metallic electrode, possessing a rapid response to changes in concentration of a given species, coupled to a standard reference electrode. Consider a system containing a solution of an organic compound in a cell containing an inert platinum electrode and assume that initially there is no oxidizing agent present. The surface of the platinum electrode will assume a potential, the magnitude of which depends on the tendency of the ccmpound to remove or yield electrons to this electrode. With organic compounds there is little tendency for either process to occur and, as a result, the potential of the system will be rather small. Addition of an oxidizing agent to the system will result in electron withdrawal from either the organic ocmpound or from the platinum electrode, depending on whether or not oxidation occurs. If oxidation does not proceed the electrons cannot be supplied from the organic compound and, therefore, the oxidizing agent must remove them from the platinum electrode which therefore, immediately assumes a large positive potential. If, on the other hand, oxidation does occur, electrons will be preferentially supplied by the organic compound and the potential of the system will not be markedly affected as its value will be determined either by the reduced form of the oxidant or by the oxidized organic canpound. As oxidation proceeds there will be little variation in potential until the equivalent point is reached, thereafter further addition of oxidant will result in a rapid increase in potential corresponding to the unmodified oxidant. '!'he described behaviour has been illustrated in Figure 2.1 for - 41 - the potentianetric titration of an organic compound by an oxidant undergoing the reversible reduction, 1m7+ + 5E. ) Mn2+ Application of the method for investigating the oxidation of amino acids. Drake and Smythe75 employed the potentiometric technique for investigating the oxidation of tyrosine, tryptophan and phenylalanine by potassiwn permanganate and eerie anmoniurn sulphate, but were able to achieve an end-point only with tyrosine oxidized by eerie ammonium sulphate. Their failure with the other systems may be attributed to the fact that they restricted their investigation to the addition of only four equivalents of oxidizing agent. The possibility of using this method for studying the oxidation of the amino acid constituents of wool was, therefore, examined. At the outset it was considered necessary to limit the investigation to those constituents particularly labile to oxidation. An indication of oxidation-lability can be easily obtained by determining the rates at which standard solutions of the amino acids react with a typical oxidizing agent. Potassium permanganate is ideally suited for this purpose, as it has been used extensively to oxidize woo137,100 and since its reduction is accompanied by a decrease in its potential and by a loss of its characteristic purple colour. The rates at which the potential of the permanganate decreases FIG. 2. 1 - Potentiometric titration curve for the oxidation of an organic compound by permanganate ion.

0.8

Mn------7+ 0.7

U) ...... 0.6 0 > .... ~ ...... i:: ....Q) 0 0.5 ~

0.4

Mn2+

0.3

0 1.0 2.0 3.0 4.0 Volume of oxidant added (mls.) - 42 - and at which the decolourization occurs should, therefore, give an indication of the ease of oxidation of the amino acids. The results of this determination indicated that the majority of amino acids required periods ranging from several hours to a few days to achieve the required decrease in potential and decolourization of the permanganate. The exceptions were cystine, cysteine, methionine, tyrosine and tryptophan, all of which decolourized the permanganate within a few minutes at room temperature. This appears to contradict the claim made by Alexnader, Carter and Hudson82 that only cyetine and tyrosine react rapidly with permanganate but these workers do not give any indication of their analytical method. The subsequent investigation was limited to these five amino acids. Not all oxidizing agents may be employed for potentio­ metric titrations, the criterion for applicability being that the oxidant must produce a significant increase in potential when added to a solution of a non-oxidizable acid such as sulphuric acid. Of the oxidizing agents potassium permangan- ate, sodium hypochlorite, sodium pereulphate, peracetic and permonoeulphuric acids, only the first two were found to satisfy the above criterion. The subsequent investigation, therefore, was confined to the use of these two reagents as oxidants. Experimental procedure. Initial experiments showed that mixtures of the reactive - 43 - amino acids and the oxidant imparted a charge to the platinum electrode but the potential drifted rapidly with time. This behaviour was also noted by Drake and Smythe75 who found that the most stable potentials were obtained in strongly acid solutions. It was found that as long as the pH of the solution was 2 or less, adequate stabilization of the potential was obtained. The solutions of the oxidizing agents were all decinormal, the solvents being sulphuric acid (0.5N) and distilled water for potassium permanganate and sodium hypochlorite respectively. The exact normalities of the oxidizing agents were ascertained by the procedures described in Voge1104• Standard solutions (O.OlM) of the amino acids in sulphuric acid (0.5N) were prepared. In the case of cystine it was necessary to employ a small amount of concentrated sulphuric acid to achieve complete dissolution. A 2.5 ml. aliquot of an amino acid standard solution was transferred to a glass cell by means of a graduated micro­ pipette and this was diluted to 25 ml. by addition of 0.5N sulphuric acid. The cell was then placed in a water-bath maintained within !0.1°0 of the desired temperature by a thermostatic heater. The sample was allowed to equilibrate tor one hour while a stream of oxygen-free nitrogen was passed through it to remove dissolved oxygen. After equilibration the potential of the system was measured by means of a - 44 - potentiometer (Radiometer Titrator type TTT le) employing a platinum electrode (Radiometer electrode type PlOl) coupled to a standard reference calomel electrode (Radiometer electrode type K4O1). The titration was commenced by adding oxidant with a micro-syringe (Hamilton syringe 725-NCH incorporating a Chaney adaptor), the solution being continuously stirred by the passage of oxygen-free nitrogen and readings of the potential were made just before subsequent additions of the oxidant. The time interval between successive additions of oxidants depends largely on the rate of reaction between the oxidant and the particular amino acid being investigated. This was arbitrarily chosen as the time required for the potential of the system to approach the initial potential after the first addition of oxidant. The Oxidation of the Amino Acids (1) The oxidation of cystine. (a) Oxidation by potassium permanganate. The results of the potentiometric titration of cystine by permanganate are illustrated by Figure 2.2. Evidently from these results, the oxidation of cystine by acidified potassium permanganate is extremely canplex. The initial addition of permanganate to cystine results in the decolourization of the oxidant within one minute but this is accompanied by an increase in the potential of the system. FIG. 2. 2 - Potentiometric titration curve for the oxidation of cystine by potassium permanganate. 0 Temp. 31 C. 1. 1

1.0

0.9

0.8

-Cl) ..-4.... 0 -> ..-4 ...... n! 0.7 i::: ....Q) 0 fli

0.6

0.5

0.4

0 1.0 2.0 3.0 Volume of O. lN potassium permanganate (mls.) (a) Oxidant added at 7 min. intervals. (b) Oxidant added at 10 min. intervals. (c) Oxidant added at 17 min. intervals. (d) Oxidant added at 26 min. intervals. - 46 -

The potential rises rapidly to a value of approximately 100 mv less than that for u.nreacted permanganate and it remains at this value for nearly three minutes when it corrmences to decrease slowly. From the rate of permanganate decolouriza- tion it would appear that it reacts extremely rapidly with cystine, but the potentiometric data indicate that the reaction proceeds in stages and the complete reduction of permanganate to the manganous state requires considerable time. These phenomena may be explained by the following reaction scheme: The first stage of the reaction between cystine and permanganate involves the formation of an unstable complex (I) which innnediat.ely gives rise to another complex (II) between manganese (III) and a partially oxidized derivative of cystine,

R R \ \. s o-...... ✓,O s +a, ~o + Mn (VII) Mn (VII ! o..,. """'o i ➔ o.,.... ""'-o / / R R

R \. s - I s - / R II

This explains both the rapid decolourization of the oxidant - 46 - and the increase in the potential of the system. It has been auggested105 that the complex is formed by coordination through oxygen atoms to the manganese (III) resulting in the potential of the system being somewhat lower than that tor unreduced permanganate. The second stage of the reaction is the relatively slow decomposition of the complex to an intermediate oxidation product of cystine and to manganic ions. The potential does not decrease significantly at this stage since the manganese remains at the same oxidation level. The third stage of the oxidation is the slow reduction of manganese (III) to manganese (II) and this would be accompanied by the slow decrease in potential toward its initial v~lue• .Additional evidence for the complex-mechanism was obtained by a spectrophotometric study of the colour changes occurring during the oxidation. These experiments were carried out qualitatively in 6N sulphuric acid in order to employ concentrated solutions of cystine thereby rendering the colour changes produced more discernible. The addition ot permanganate to cystine results in the decolourization of the oxidant to an unstable yellow colour which rapidly changes to a piuk-red. The yellow colour is thought to arise from the formation of a manganese (III) complexl05 and the absorption spectrum of this species is shown in Figure 2.3(a). The spectrum was obtained by employing very dilute solutions - 47 - of cystine and permanganate (O.OOlM and O.lN respectively both at pH2) so that the decolourization of the yellow species would be decreased to such a rate that a spectral record could be obtained. The pink-red solution is thought to arise from the decomposition of the complex to yield uncomplexed manganic ions and the spectral absorption of this solution is shown in Figure 2.3(b). It is known that manganous ions can be oxidized to manganic by potassium permanganatel06 and this was carried out to obtain the spectrum of the resulting pink­ red solution shown in Figure 2.3(c). Clearly, this cannot be caused by unmodified potassium permanganate since its spectrum, shown in Figure 2.3(d), is entirely different. It is evident that the spectra presented in Figures 2.3(b) and (c) are the same and therefore, it is very likely that they arise from the same species in solution, namely manganic ions. On standing, the pink-red solution gradually becomes colourless, corresponding to the reduction of manganic ions to manganous ions. Clearly the spectral evidence is in good agreement with the proposed reaction scheme. The actual nature of the manganese (III) - cystine complex has not been elucidated and several possible structures may be proposed: FIG. 2. 3 - Visible spectra of coloured species produced by oxidation of cystine with potassium permanganate.

0.8

0.6 ...... >- ID i:: Q) re, 0.4

RI -....u ...p.. 0 0.2

0 0

Wavelength (m,?-)·

(a) Yellow solution.

(b) Pink-red solution.

( c) Permanganate oxidized manganous ions.

(d) Potassium permanganate. - 48 -

3+ R R R '\ '\ I a-') o~ --o S ~ 0~ cO+- S I Mn (IIIL_ I Mn(III) I S---+ o_;,t 0 5_.,.QJ ,O+-S / I '\ R R R III IV CH2-SO~ O ~ . Mzf(1II) J' S04-- H3Ni°-CH--COOH "'o

V The eyrmnetrical cystine -s,s-I dioxide should result from structures III and IV whereas structure V would yield alanine sulphinic acid. Alanine sulphinic acid is known to decompose slowly and on the basis of the known instability of other partial oxidation products44 of cystine it is likely that cystine -s,s~ dioxide would also hydrolyse readily. The fact that s,s~ dioxides of proven structure are unknown4 4 favours Vas the likely structure of the complex but the evidence is not unequivocal. Additional evidence presented later in this and the subseg.,.ent chapter, signifies that the intact disulphide bond is a necessary requirement for the formation of the complex, so that it is more likely that the complex possesses a synnnetrical s,s-I dioxide configuration. In order to obtain an estimate of the number of equivalents

or permanganate required to oxidize cystine by the procedure or Drake and Smythe75, it is necessary to employ the 26 minute- - 49 - interval plot shown in Figure 2.2, for it is clear that the reduction of permanganate to manganous is virtually ccmplete after this time. Unless the oxidation is nearly spontaneous, it is extremely difficult to assess accurately the end-point of the titration and this is particularly so for the permanganate-cystine system. Two approaches may be used for this estimation. In the first approach, the titration curve is taken to be ideal and the point of inflection then represents the end- point. Applying this to Figure 2.2, the end-point appears to be reached on the addition of ten equivalents, which is the theoretical amount for the quantitative conversion of cystine to cysteic acid. Since cysteic acid is the final product of cystine oxidation, it would appear that this approach is reasonable but, since the system is far from ideal, this result must be treated with some reservation. The second approach assumes that the oxidation is complete at the point where the large increase in potential occurs. The necessary condition is that oxidation should be complete between successive additions of oxidant, for if this is the case, then the reaction should proceed at a fairly constant rate resulting in uniform variations in potential. As soon as the oxidation reaches completion there should be a marked increase in potential resulting in a distinct discontinuity of the titration curve. The end-point of the titration may - 60 - be obtained by extrapolating both parts of the curve before and during the rapid rise in potential and the point of intersection then corresponds to the end-point. It is thought that the latter method for the estimation of the end-point of the oxidation is the more reliable, since the system under consideration is not ideal and appears to occur in separate stages. Applying this approach, the end­ point of the cystine-permanganate titration appears to occur at the addition of approximately eight equivalents of oxidant. This value corresponds to the addition of four atoms of oxygen and possibly denotes the formation of cystine-S,S- disulphone, 4(0] Cy.S.S.Cy The increase in potential on the addition of more than eight equivalents of oxidant is thought to arise from the slower oxidation of the disulphone to cysteic acid. (b) Oxidation by chlorine. The results of the potentiometric titration of cystine by chlorine are shown in Figure 2.4(a). The oxidation of cystine by chlorine occurs very rapidly as evidenced by the almost innnediate attainment of the original potential of the system, after the initial increase caused by the addition of the oxidant. Thie behaviour is in marked contrast from that observed when permanganate is employed as the oxidant. It would appear, therefore, that chlorine effects the complete FIG. 2. 4 - Potentiometric titration curves for the oxidation of cystine. 3 min. intervals between successive additions of oxidant. Temp. 31°C.

1. 1

1.0

0.9

0.8 ...,(I) -0 -> -...... ,nl ~ 0.7 ...,Q,) 0 ~

0.6

0.5

0.4 0 l. 0 2.0 3.0 4.0 Volume of 0. lN oxidant (mls.) (a) Chlorine.

(b) Potassium permanganate-sodium chloride - 51 - oxidation of cystine more rapidly than permanganate under identical conditions of treatment. From Pigure 2.4(a) it is clear that the titration end­ point is reached on the addition of approximately ten equivalents of oxidant and this corresponds to the conversion of cystine directly to cysteic acid, 6 [OJ Cy.S.S.Cy

(ii) The oxidation of cysteine. (a) Oxidation by potassium permanganate. The results of the potentiometric titration of cysteine by potassium permanganate are illustrated in Figure 2.5(a). The main feature of the oxidation of cysteine by permanganate is the spontaneous decolourization of the oxidant, accompanied by the almost instantaneous attainment of the initial potential of the system. The oxidation appears to proceed at a relatively uniform rate up to the addition of four equivalents of oxidant but on the addition of another equivalent of oxidant the reaction rate decreases as evidenced by the increase in potential and this corresponds to the titration end-point. The above data cannot be resolved on the basis of initial disulphide formation arising from the oxidation of a thiol, for this would require the addition of only one equivalent of oxidant and the subsequent oxidation would be expected to FIG. 2. 5 - Potentiometric curves for the oxidation of cyst eine. 3 min. (a) 1. 1 intervals between successive additions of oxidant. Temp. 31°C.

1. 0

0.9

0.8

-...... Ill 0 > 0.7 -.... c1S ...... I:: ....Q) 0 ~ 0.6

0.5

0.4

0.3 0 1. 0 2.0 3.0 4.0 5.0 Volume of 0. 1 N oxidant added (mls . ) (a) Potassium permanganate. (b) Chlorine. (c) Potassium permanganate-sodium chloride. - 52 - proceed in an analogous manner to that of cystine and clearly, this does not occur. The potentiometric data may be explained on the basis of the following reaction scheme: (1) The first stage involves the formation of alanine sulphinic acid corresponding to the addition of two oxygen atoms to the cysteine, i.e., four equivalents of oxidant, Cy. S. H 2 [O] > Cy. S02• H (11) The second stage requires the addition of one equivalent of oxidant and therefore, appears to involve the elimination of the hydrogen atoms to form cystine disulphone, Cy. 802• H + Cy. S02• H - 2 [H]> Cy. S02• 802• Cy From these results it is evident that the oxidation of cysteine by permanganate does not involve the formation of a disulphide bond and clearly, the oxidation of this thiol does not proceed through the formation of an intermediate complex with manganese (III). These observations, therefore, indicate that the presence of an intact disulphide linkage is probably a necessary requirement for the formation of the complex between manganese {III) and a partially oxidized cystine derivative. On this basis it is, therefore, possible to eliminate proposed structures for this complex based on initial disulphide breakdown. Pu.rther confirmation concerning the non-formation of the disulphide during the oxidation of cysteine was obtained polarographically and is presented in - 53 - the following chapter. (b) Oxidation by chlorine. The results of the potentiometric titration of cysteine by chlorine are shown in Figure 2.5(b). The oxidation of cystei~e by chlorine proceeds very rapidly since the potential of the system instantly regains its original value on the addition of chlorine. From Figure 2.5(b) it is evident that the titration end-point corresponds to the addition of nine equivalents of chlorine. The oxidation of cysteineto cysteic acid requires only six equivalents of oxidant and since the potentiometric data indicate that more oxidant is necessary, it appears that the oxidation proceeds to a higher level involving oxidative degradation of cysteine. In view of the fact that cystine is oxidized directly to cysteic acid by chlorine, the result for cysteine is inexplicable and further examination of this problem is necessary. (111) The oxidation of methionine. (a) Oxidation by potassium permanganate. The results of this determination are illustrated in Figure 2.6(a). The potentiometric data indicate that the oxidation of methionine by permanganate is extremely rapid, the titration curve being characterized by a marked discon­ tinuity after the addition of two equivalents of oxidant.

The oxidation of methionine, therefore, appears to proceed rapidly with the formation of the sulphoxide, FIG. 2. 6 - Potentiometric titration curves for the oxidation of methionine. Temp. 31°C. (a)

1.0 (c)

0.9

0.8

0.7

en ...... 0 > -..... 0.6 ....ell ...s:: ...(I) 0 0..

0.5

0.4

0.3

0 1. 0 2.0 3.0 Volume of 0. 1 N oxidant added (mls. ) (a) Potassium permanganate ( 4 mins. oxidant addns.). (b) Chlorine (2 mins. oxidant additions). (c) Potassium permanganate-sodium chloride (2 mins. oxidant additions). - 54 -

Oxidation of methionine to the sulphone stage is also known10l but the potentiometric data indicate that addition of the second oxygen atom proceeds at a slower rate under the conditions employ~d here. (b) Oxidation by chlorine. The results of the potentiometric titration of methionine by chlorine are shown in Figure 2.6(b). The oxidation of methionine proceeds very rapidly and from Figure 2.6(b} the potentiometric end-point appears at the addition of seven equivalents of chlorine. The oxidation of methionine to methionine sulphone requires only four equivalents of oxidant and since an additional three equivalents are needed it is likely that oxidative degradation of methionine sulphone subsequently occurs. (iv) The oxidation of tyrosine. (a) Oxidation by potassium permanganate. The results of the potentiometric titration of tyrosine by potassium permanganate are shown in Pigure 2.7(a). Tyrosine oxidation by permanganate proceeds very rapidly as evidenced by the virtual instantaneous decolourization of the oxidant and by the very swift decrease in potential after the initial increase caused by the addition of the oxidant. These observations indicate that the permanganate is being reduced directly to the manganous state and the resulting - 55 - yellow solution is probably associated with the oxidized derivatives of tyrosine and not with any manganese (III) complex. The titration curve shown in Figure 2.7 (a) exhibits a marked discontinuity at a point corresponding to the addition of eight equivalents of permanganate, subsequent additions of oxidant causing the potential to increase sharply. It is unlikely that oxidative degradation of the alkyl side chain of tyrosine occurs, since permanganate has no action on phenylalanine under these conditions and this amino acid differs from tyrosine only by the absence of the phenolic hydroxyl group. It is more likely that the aromatic ring is affected but it is extremely difficult to formulate a compound arising from tyrosine by its interaction with eight equivalents of oxidant, except on the basis of ring degradation. Indole formation due to cyclization of the alkyl side chain of tyrosine during oxidation has been postulated by a number of workers76, 81 but such compounds have not been isolated from tyrosine oxidized by permanganate. Furthermore, the oxidation by permanganate of tyrosine in the free and combined states appears to be very similar (Chapter lV) and since the alkyl side chain is not free to cyclize when in the combined form, it would appear that indole formation does not occur under the conditions employed. The formation of a para-quinone has been postulated as a FIG. 2. 7 - Potentiometric titration curves for the oxidation of tyrosine. 3 min. intervals between successive addition of oxidant. Temp. 31°c.

1 . 1

1.0

0.9

0.8

...,(ll -0 -> Ill -...... , 0.7 ~ ...,Q) 0 p..

0.6

0.5

0.4 0 1. 0 2.0 3.0 4.0 5.0

Volume of 0. 1 N oxidant added (mls.) (a) Potassium permanganate. (b) Chlorine. (c) Potassium permanganate-sodium chloride. - 66 - reaction product of the oxidation of tyrosine by permanganate82 but this would require only two equivalents of oxidant which is incompatible with the experimental result. It is possible for hydroxylation of the aromatic ring to occur with the formation of dihydroxyphenylalanine (dopa) and for this to be converted into the ortho-quinone as shown, ~COOH COOH [01 • ~c~) ,,. HOV JH2. OH 2 dopa ortho-quinone This would also require less oxidant than is observed experimentally. In order to account for the additional oxidant it is necessary to postulate the disruption of the aromatic ring leading to the formation of products such as cis, cis-muconic acid107, oxalic acid and ultimately to carbon dioxide. This is not unreasonable since it is known that permanganate can disrupt the aromatic ring of toluene under acidic conditions108• (b) Oxidation by chlorine. The results of this determination are shown in Figure 2.7(b). The oxidation of tyrosine by chlorine proceeds rather slowly as evidencsiby the fact that the potential does not decrease to the original value in the time interval employed between successive additions of oxidant. Moreover, further addition of oxidant to the system causes the potential to increase markedly in contrast to the behaviour observed when potassium - 57 - permanganate is employed as the oxidant. Clearly from Figure 2.7(b) it is not possible to assign a potentiometric end-point for the oxidation of tyrosine by chlorine so that no indication of the possible product can be obtained. However, from the known reaction of chlorine with tyrosine, it is likely that the eventual product is 3,5- dichlorotyrosine76 but it would appear that under the above experimental conditions this is formed only slowly. (v) The oxidation of tryptophan. (a) Oxidation by potassium permanganate. The results of the potentiometric titration of tryptophan by acidified potassium permanganate are shown in Figure 2.a(a). Tryptophan combines very rapidly with permanganate since the oxidant is decolourized almost immediately to a yellow solution and since the initial potential of the system is rapidly regained after the addition of oxidant. These results suggest that the permanganate is reduced directly to manganous ions. From Figure 2.a(a), the potentiometric curve exhibits a discontinuity at a point corresponding to the addition of fourteen equivalents of permanganate, assuming a five electron reduction of manganese (VII) to manganese (II). The large number of equivalents of permanganate necessary to oxidize tryptophan possibly indicates that both ring disruption and substitution occurs and, therefore, an assignment of a possible FIG. 2. 8 - Potentiometric titration curves for the oxidation of tryptophan. 3 min. intervals between successive 0 additions of oxidant. Temp. 31 C. (b)

1.0

0.9

0.8

Cl) 0.7 ....+-' 0 > -...... nl +-' s:: 0.6 ~ +-' 0 0.

0.5

0.4

0.3 _....____...... __._..__.....__.__...,____...... __._...___._..__ ...... _..__ ...... 1-...... __._..._ ..... _ 0 1.0 2.0 3.0 4.0 5.0

Volumes of 0. lN oxidant added (mls.) (a) Potassium permanganate. (b) Chlorine. (c) Potassium permanganate-sodium chloride. - 68 - reaction scheme is extremely difficult. A chromatographic analysis of this system (Chapter IV) indicates that the pyrrole ringCl:::Jl:COOH is split to form kynurenine which possibly arises as shown, N NH2 [O] NMa . H >

lcynurenine

However, because of the complexity of this reaction it was not pursued further in the present investigation. (b) Oxidation by chlorine. The results of the potentiometric titration of tryptophan by chlorine are shown in Figure 2.S(b). It is clear from the potentiometric curve that tryptophan does undergo an initial, rapid oxidation by chlorine and this is complete on the addition of approximately four equivalents of oxidant. It would appear that the oxidation causes the disruption of the 2:3 carbon-carbon bond of the pyrrole ring, resulting in the formation of a substituted kynurenine derivative,

~COOH

~ ...~ ) Jw_NH~ _ _;;_;;;,__[Q}

Clearly, from the slope of the potentiometric titration curve, a second oxidation reaction must be proceeding at a much slower rate than the initial oxidation involving the addition of four equivalents of chlorine. This probably - 59 - corresponds to a slower substitution reaction of the aromatic ring but this could not be demonstrated directly.

The Oxidation of the Amino Acids in the Presence of Sodium Chloride by Acidified Potassium Permanganate The presence of certain saturated salt solutions have been found to exert a protective effect on wool during alkali or oxidation treatments109,lOO. In particular, the oxidation of wool in concentrated solutions of sodium chloride by potassium permanganate has received some attention37,lOO. The maixi residue affected in this treatment is cystine although damage to serine also occurs37• No significant losses of tyrosine nor tryptophan have been reported during these treatments, a surprising result when their reactivity with permanganate alone is considered. Although there are some differences in the chemical modifications produced in wool by oxidation with permanganate in the presence or absence of salt, it has been suggested that the added salt functions only to change the site of the reaction, so that with increasing salt concentration the amount of fibre surface modification also increasesllO. The effect of neutral potassium permanganate on the free amino acids in saturated sodium chloride has not been investigated, since these amino acids are relatively insoluble under neutral conditions. However, it was decided - 60 - to investigate the oxidation of the free amino acids in acidified solutions of sodium chloride by potassium permanganate in order to determine whether the chloride ions play a passive or active role in the oxidation. The same experimental proced~e was employed as described above, except that in this case 6g. sodium chl_oride/25 ml. of solution were added. (1) The oxidation of cystine. The results of the potentiometric titration of cystine in concentrated sodium chloride solution by acidified potassium permanganate are presented in Figure 2.4(b). The oxidation of cystine by acidified potassium permanganate is greatly influenced by the presence of sodium chloride. It is faster in the presence of sodium chloride as evidenced by the immediate decolourization of the permanganate and by the very rapid attaimnent of the initial potential of the system on addition of the oxidant. It was also found that decolouriza- tion of permanganate occurs when it is added to a solution of sodium chloride alone but, in this case, the potential increases and this is thought to arise from the oxidation of chloride to chlorine by permanganate. The fact that the potential of the system drops rapidly toward its initial value when permanganate is added to cyetine in concentrated salt solution, indicates that the cyetine undergoes oxidation very quickly and that the oxidant is reduced to manganous ions. Since both cystine and chloride - 61 - ions can be oxidized separately by permanganate, the primary oxidation step involves either the oxidation of chloride to chlorine, which then rapidly oxidizes the cystine, or the oxidation of cystine, with the salt somehow functioning to accelerate the oxidation of cystine, and clearly, the evidence presented here is inadequate to distinguish between these two mechani ems. Additional evidence, to be presented later in Chapter III, indicates that the oxidation is not a chlorination reaction, but since the salt must play an active role in the oxidation of cystine by permanganate it must function in one of two ways. Firstly, it may hasten the decomposition of the manganese (III) complex or secondly, the salt may prevent the oxidation from proceeding by this mechanism and consequently cause a rapid reduction of the permanganate straight to manganese (II). To obtain some information concerning the role of the salt in this system the following experiment was carried out. The oxidation of cystine by permanganate was investigated potentiometrically as described in part 2 but here, after the formation of the manganese (III) complex (as evidenced by the yellow solution and the elevated value of the potential) a saturated solution of sodium chloride was quickly added. This addition resulted in a rapid decolourization of the solution and a rapid decrease in the potential of the system, - 62 - indicating the swift decomposition of the complex with a simultaneous reduction of the manganese (III) to manganese (II). This procedure was repeated with saturated solutions of potassium bromide, sodium nitrate and sodium sulphate but only the former was found to behave in a similar manner to sodium chloride. Apparently, therefore, the halide ions are capable of disrupting the manganese (III) complex and of causing the rapid reduction of manganese (III) to manganese (II), in agreement with various inorganic complexes of manganese (III). This result, however, does not eliminate the possibility that the halide ions prevent the manganese (III) complex from being formed entirely, thereby causing the permanganate to be reduced directly to manganous ions. However, these results do indicate that the presence of high concentrations of halide ions modifies the oxidation reaction between cystine and per­ manganate and the possibility that this effect also occurs in the oxidation of wool keratin in brine by permanganate should not be overlooked. (11) The oxidation of cysteine. The results of the potentiometric titration of cysteine in salt solution by potassium permanganate are shown in Figure 2.6(c). The presence of sodium chloride does not appear to have a significant effect on the oxidation of cyateine by permanganate. The oxidation is extremely rapid - 63 - and from Figure 2.5(c) it appears to be completed on the addition of five equivalents of oxidant and this indicates that a possible reaction product is cystine disulphone. (iii) 'l'h.e oxidation of methionine. The results of the potentiometric titration of methionine in concentrated sodium chloride solution are shown in Figure 2.6(c). The presence of sodium chloride appears to accelerate the oxidation of methionine by permanganate and this was evidenced by a more rapid decrease in the potential toward the original potential on addition of the oxidant. The oxidation appears to be complete on the addition of two equivalents of permanganate and this corresponds to the formation of methionine sulphoxide. Further oxidation of the sulphoxide to the sulphone stage probably proceeds at a decelerated rate. (iv) The oxidation of tyrosine. The results of the potentiomnetric titration of tyrosine in saturated salt solution by potassium permanganate are shown in Figure 2.7(c). It is clear that the oxidation of tyrosine in the presence of salt proceeds quite rapidly (almost as rapidly as in the absence of salt) as evidenced by the rapid decolourization of the oxidant and the swift attainment of the original potential of the system after the initial increase due to the added unreacted oxidant. A comparison of Figures 2.7(a) and 2.7(c) indicates that the salt does modify the - 64 - oxidation of tyrosine and this can be seen from the fact that the amount of oxidant necessary to obtain the titration end­ point in the presence of salt is less than when salt is excluded. The rapid rate of oxidation of tyrosine in salt by permanganate indicates that the reaction is not a chlorination. It is thought that, up to the addition of six equivalents, the oxidation in salt proceeds in a similar fashion to that when salt is excluded from the system. At this stage it is likely that the oxidation of chloride ions to chlorine by permanganate requires less energy than the subsequent oxidation of tyrosine and, therefore, the former reaction occurs, resulting in the observed potentiometric end-point caused by unreacted chlorine. (v) The oxidation of tryptophan. The results of the potentiometric titration of tryptophan in saturated salt by permanganate are shown in Figure 2.8(c). The presence of salt appears to have a similar effect on the oxidation of tryptophan as observed for that of tyrosine. The salt here is thougbt to function in an anologous manner to that observed for tyrosine, in that it prevents the oxida­ tion of tryptophan from proceeding to the same level attained when salt is absent. - 65 -

The Oxidation of Soluble Proteins (1) The oxidation of insulin. The potentiometric procedure was next employed to study the oxidation of soluble proteins and in particular to the invest~gation of the oxidation of insulin. Insulin is a particularly useful model for investigating the oxidation of the combined amino acids cystine and tyrosine, since these are present in approximately the same percentages and further insulin does not contain the other oxidation-susceptible amino acids tryptophan, cysteine and methionine. The experimental procedure employed for this determination was similar to that described in part 2 for the free amino acids, except that here 20 ml. aliquots of insulin (0.1% in pH2 modified universal buffer consisting of phosphoric, acetic and boric acids) were titrated potentiometrically with potassium permanganate and chlorine. (a) Oxidation by potassium permanganate. The results of this determination are shown in Figure 2.9(a}

The oxidation of insulin proceeds very rapidly as evidenced by the swift decrease in the potential corresponding to the unreacted oxidant. Evidently from Figure 2.9(a) the potential shows a marked discontinuity at the addition of 1.25 ml. of oxidant. From the results of part 2 it is possible to calculate the amount of permanganate required to oxidize separately the combined tyrosine and cystine in 20 ml. of O.l~ FIG. 2. 9 - Potentiometric titration curves for the oxidation of insulin and free amino acids corresponding to insulin by potassium permanganate. 3 min. intervals between successive additions of oxidant. Temp. 31°C.

1.1

1.0

0.9

0.8

fl) +' ..-4 0 -> ..-4 0.7 ....RI +' s::: GJ +' 0 ~

0.6

0.5

0.4 0 1.0 2.0 3.0 4.0 Volume of 0. lN potassium permanganate added (mls.) (a) Insulin.

(b) Amino acids corresponding to insulin. - 66 - insulin and this corresponds to 1.3 and 0.83 ml. of oxidant respectively. If the oxidant reacts with both residues simultaneously the amount of oxidant required should be 2.13 ml. The experimental result is in good agreement with that necessary for the preferential oxidation of combined tyrosine but because of lacking supporting evidence the result is not unequivocal. It was decided to titrate potentiometrically a mixture of the free amino acids in the same proportions as their occurrence in 20 ml. of 0.1% insulin with potassium permanganate and these results are shown in Figure 2.9(b). Clearly, the potentiometric titration curve exhibits a marked discontinuity at precisely the same amount of added oxidant as required for the titration of insulin itself. It would appear, therefore, that the oxidation of the free and combined amino acids are very similar and since the amount of oxidant employed is exactly the same required for the preferential oxidation of tyrosine, it is thought that permanganate effects the prefer­ ential oxidation of free and combined tyrosine from mixtures of it am cystine. This result is supported by additional evidence presented in the following chapters. (b) Oxidation by chlorine. The results of the potentiometric titration of insulin by chlorine are illustrated by Figure 2.10 (a). The titration curve exhibits a marked discontinuity at the addition of - 67 -

1.26 ml. of oxidant. Because of the slow rate of oxidation of tyrosine by chlorine it is thought that the chlorine may react preferentially with the combined cystine. On this basis the amount of chlorine required should be approximately 1.04 ml, based on the oxidation of free cystine by chlorine (part 2). The fact that the observed and calculated amounts of oxidant differ indicates that chlorine does not oxidize the cystyl residues of insulin specifically. The potentiometric titration by chlorine of an amino acid mixture corresponding to 20 ml. of 0.1% insulin was carried out and the results are illustrated by Figure 2.lO(b). In this case the titration curve shows a marked discontinuity at the addition of approximately 1.0 ml. of oxidant, in fair agreement with the calculated value of 1.04 ml. These results signify that chlorine differs slightly in its attack on free and combined amino acids and apparently some other group in insulin is being attacked by this oxidant. The above results indicate that selective oxidation of tyrosine or cystine, bQth in the free or combined states, may occur by employing either potassium permanganate or chlorine respectively. This behaviour has been previously reported for these amino acids in the free state but unfortunately no experimental details were presentedlll. Because of this and because the evidence based on potentiometry alone can only be considered as tentative, it was decided to investigate in FIG. 2. I 0 - Potentiometric titration curves for the oxidation of insulin and free amino acids corresponding to insulin by chlorine. 3 min. intervals between successive additions of oxidant. Temp. 31°c. I.I

1.0

0.9

...fll ~ 0 -> 0.8 ~ ....nl ~ Q) ...0 ~ 0.7

0.6

0.5 0 1.0 2.0 3.0 4.0

Volume of 0. 1 N chlorine added (mls. ) (a) Insulin. (b) Amino acids corresponding to insulin. - 68 - detail the oxidation of cystine by potassium permangante and chlorine in the presence or absence of tyrosine by a polaro­ graphic procedure. This investigation is described in detail in the following chapter. (11) The oxidation of lysozyme. Lysozyme provides a useful model to study the reactivities of combined cystine, tyrosine and tryptophan and the percentage contributions of these residues to the total amino acid composition of lysozyme are 14.2, 3.7 and 8.3% respectively112• The experimental procedure employed here was essentially the same as that described for insulin except that here 25 ml. aliquots of lysozyme (0.1% in pH2 modified universal buffer) were titrated with potassium permanganate and chlorine. (a) Oxidation by potassium permangante. The results of this determination are shown in Figure 2.ll(a). The oxidation of lysozyme by potassium permanganate proceeds very swiftly as evidenced by the rapid decrease in the potential corresponding to the unmodified oxidant. Fran Figure 2.ll(a) it is evident that the titration curve exhibits a marked discontinuity at the addition of 1.5 ml. of oxidant. It is possible from the results of part 2 to calculate the amount of permanganate required to oxidize separately the combined tyrosine, cystine and tryptophan in 25 ml. of 0.1~ lysozyme (assuming that the free and combined amino acids react in the same way) and this corresponds to 0.47, 1.34 and FIG. 2. 11 - Potentiometric titration curves for the oxidation of lysozyme. 3 min. intervals between successive additions of oxidant. Temp. 31°C.

0.9

Ul ...... 0 > ..... Cl! ...... 0.8 i:: ...ii) 0 ~

0 1.0 2.0 3.0 4.0 5.0

Volume of 0 . 1 N oxidant added (ml s . ) (a) Potassium permanganate. (b) Chlorine. - 69 -

1.46 ml. of oxidant respectively. It is clear from the experimental end-point that the oxidant does not react with all three amino acid residues completely since this would entail the use of 3.27 ml. of added oxidant to achieve the end-point. It is, therefore, possible that either the amino acids react differently in the combined state from the free state or there is some form of preferential oxidation occurring. From the fact that the calculated amount of oxidant required to oxidize the combined tryptophan in lysozyme agrees so closely with the observed end-point, it is thought that the permanganate reacts prefer­ entially with the tryptophyl residues of lysozyme. This result is supported by additional evidence presented in Chapter IV. (b) Oxidation by chlorine. The results of this determination are shown in Figure 2.ll(b). It is evident that the potentiometric end-point occurs at the addition of 2.26 ml. of oxidant. The amounts of oxidant required to oxidize combined cystine and tryptophan in 25 ml. of 0.1% lysozyme, calculated from the results of part 2, are 1.36 and 0.41 ml. respectively. Because of the somewhat sluggish reaction between tyrosine and chlorine, it 1s not possible to obtain an estimate of the amount of chlorine required to oxidize this residue in lysozyme. It is clear that the combined volume of oxidant calculated to oxidize both - 70 - cystine and tryptophan (1.77 ml.) is considerably lower than the experimental value and as a result it is thought that, apart from these residues, some other group in the protein lysozyme undergoes modification by the chlorine. This behaviour appears to be analogous to that observed for the oxidation of insulin by chlorine.

Conclusion The preceding potentiometric investigation has yielded data suggesting that oxidation of mixtures of amino acids in the free state or combined in proteins may occur in preferen­ tial stages. In order to examine further this suggestion, it was decided to study the oxidation of mixtures of free and protein-bound amino acids by the techniques of polarography and ultraviolet difference spectroscopy. - 71 -

Chapter III: A POL.AROGRAPHIC INVESTIGATION OF THE OllDATION OF CYSTID, CYSTEINE AND TYROSINE

Introduction

Po~arography is a term applied to an electrochemical method for analyzing various inorganic and organic species and the theoretical and applied aspects of this technique have been treated in detail by a number of workers113,ll4. Essentially, polarography is based on the study of electrolysis of electroactive chemical species at dropping mercury capillary electrodes and on the interpretation of the result­ ing current-voltage curves (polarograms). A simple polarographic arrangement is shown in Figure 3.1. Consider the current-voltage curve obtained by the electrolysis of a non-electroactive species such as a borate buffer at pH9 (curve 1, Figure 3.2). As the applied potential is increased, the current flowing in the system increases only very slightly until at point D it connnences to increase very sharply. This large increase of current takes place at a very high negative potential and corresponds to the electrodeposition of sodium ions (buffer components) at the dropping mercury electrode and involves the formation of a dilute amalgam. If a dilute, oxygen-free solution of an electroreducible species, such as a O.OlM ethanolic solution of propiophenone in the same borate buffer, is electrolyzed then the current- FIG. 3. 1 - Schematical view of polarographic circuit.

G

M

F

B

D

V R

(C) capillary of the mercury dropping electrode. (V) electrolysis vessel. (S) solution to be analysed. (R) reference electrode.

(N2) nitrogen inlet and outlet. (M) mercury reservoir. (G) galvanometer. ( P) potentiometer slide contact. (DF) potentiometer. (B) storage battery, source of e.m.f. - 72 - voltage curve may be represented by curve 2 of Figure 3.2. The shape of this curve is characteristic of most electro- reuucible species. AB is termed the residual current and it is practically identical to the current obtained before addition of the polarographically active compound. At point B the potential commences to increase sharply and this corresponds to the electroreduction of the propiophenone to the corresponding 1-phenylpropylalcohol. At the point C the current no longer increases linearly with applied potential but approaches a steady limiting value at the I point ~ No further increase in current is observed at higher cathode potentials unless a second compound capable of being reduced at these potentials is present in the solution, and CD'is often termed the limiting current. The main feature of the polarographic curve, or wave as it is often referred to, that can be used to classify electro­ active compounds is the "half-wave" potential which corresponds to the potential at which the current is exactly half the limiting current. The half-wave potential is a physical constant depending on the particular chemical species employed and since it is practically independant of the concentration of the compound, it may be used for qualitative analyses. The actual height of the wave - given by the difference between the limiting and residual currents - is actually proportional to the concentration of the polarographically active species FIG. 3. 2. - Reduction of propiophenone.

C

(ii) (i) ......

A

E, V

Borate buffer pH 9.0; added: (i) O; (ii) 2 x 10-4M propiophenone, 2% per cent ethanol. - 73 - and consequently it may be used for quantitative measurements. The polarographic technique is not directly applicable to proteins, since only those proteins containing cysteine or cystine give rise to catalytic waves in solutions containing ammonium chloride and anmonia, while the addition of a salt of divalent or trivalent cobalt yields a protein "double catalytic wave". In either case, the heights of these waves are not, in general, proportional to the concentration of the protein. Cystine can be reduced reversibly in acidic media at a dropping mercury electrode11 5, the reduction being represented by the following equation,

Cy.S.S.Cy + + 2E --> 2Cy.SH Ben!Mekll5 has shown that the height of the polarographic wave of cystine is proportional to the concentration of the amino acid in solution and furthermore, this wave is unaffected by the presence of the other amino acid constituents of wool. This worker has extended the method for the analyses of cystine in hydrolysates of UDnodified and oxidized woo11161 but the results in the latter case must be treated with some reserva­ tion, since it is known that hydrolysis can result in disproportionation of partially oxidized cystine residues to cyetine itself. This, however, does not preclude the method from being employed to investigate the oxidation of free cystine since hydrolysis is not necessary in this case. - 74 -

Experimental procedure

The following experimental procedure was employed to investigate the oxidation of cystine alone and in the presence of tyrosine: 10 ml. a'iiquots of the amino acid (0.001M), dissolved in modified universal buffer of pH2, were pipetted into 25 ml. volumetric flasks and to these were added the requisite volume of decinormal oxidant. The solutions were then made up to volume with the buffer solution, mixed thoroughly and allowed to stand for the required time after which they were examined by the polarographic method described in Appendix Al.

Results (1) Oxidation by potassium permanganate. The results of the polarographic analyses of the oxidation of cystine by potassium permanganate are shown in Figure 3.3 where the cyetine wave is characterized by a half-wave potential of -0.85v. The solutions were allowed to stand for one hour before being examined polarographically but even after this time the solutions were yellow indicating that no significant decomposition of the manganese (III) complex had occurred in this time. The decrease in the height of the cyatine wave with increasing amounts of oxidant indicates the extent of cystine modification. The oxidation of tyrosine by permanganate was also FIG. 3. 3 - Potassium permanganate oxidation of cystine.

Curves: (i) 0. 00 ml.; (ii) 0. 25 ml.; (iii) 0. 50 ml.; (iv) 0. 75 ml.;

(v) 1. 00 ml., 0. IN KMnO 4 • Curves (i) - (v) from 0 volts. 200 mv/abscissa unit.

FIG. 3. 4 - Potassium permanganate oxidation of a mixture of cystine and tyrosine.

Curves: (i) 0. 00ml.; (ii) 0. 25 ml.; (iii) 0. 50 ml., 0. IN KMn04 Curves (i) - (iii) from 0 volts. 200 mv/ abscissa unit. - 76 - examined polarographically but it was found that neither tyro­ sine nor its oxidation products yield a polarographic wave in the voltage range employed. The only observable effect noted here was a alight increase in the residual current as the oxidatio.n proceeded. The method, therefore, should be applicable for investigating the behaviour of cystine in the presence of tyrosine during permanganate oxidation, thereby examining claims that with mixtures of these amino acids the tyrosine component is oxidized preferentially by permanganate. This determination was carried out on solutions of the two amino acids (each O.OOlM) by the procedure described above and the results are shown in Figure 3.4. Clearly, there is little or no variation in the height of the cystine wave during the oxidation of the mixture in marked contrast to the behaviour noted for the oxidation of cystine alone. The slight distortion of the cystine wave is thought to arise from the increasing residual current due to the oxidative deriva­ tives of tyrosine. This evidence, therefore, supports the suggestion that permanganate oxidizes tyrosine preferentially from mixtures of tyrosine and cystine. (11) Oxidation by chlorine. The results of the polarographic analyses of the oxidation of cystine by chlorine are presented in Figure 3.6. In this case the solutions were examined fifteen minutes after the introduction of the oxidant. Once more the decrease in the FIG. 3. 5 - Oxidation of cystine by chlorine

Curves: (i) 0.00 ml.; (ii) 0.25 ml.; (iii) 0.50 ml.; (iv) 0.75 ml.; (v) 1. 00 ml., 0. IN chlorine. Curves (i) - (v) from 0 volts. 200 mv/ abscissa unit.

FIG. 3. 6 - Oxidation of a mixture of cystine and tyrosine by chlorire .

Curves: (i) 0.00 ml.; (ii) 0.25 ml.; (iii) 0.50 ml.; (iv) 0.75 ml.; (v) 1. 00 ml. , 0. 1 N chlorine. Curves (i) - (v) 0 volts. 200 mv/abscissa unit. - 76 - height of the cystine wave testified to the rapid oxidation of this amino acid by chlorine. The results of the earlier potentiometric investigation ha,eahown that the rate of oxidation of cystine by chlorine is much.greater than that for the oxidation of tyrosine and it was decided, therefore, to study the behaviour of cystine in the presence of tyrosine during oxidation by chlorine. The results of this investigation are shown in Figure 3.6. A comparison of figures 3.5 and 3.6 indicates that the reduction in height of the cystine wave as a result of oxidation by chlorine is little different whether the cystine is oxidized alone or in the presence of tyrosine. This indicates that there is little interaction occurring between the tyrosine and chlorine, otherwise there would be consider­ able variation in the height of the cystine wave depending on whether tyrosine was present or not. This result indicates that, with mixtures of tyrosine and cystine, chlorine oxidizes the cystine component in a preferential manner. This behaviour is exactly the opposite noted for the oxidation of these amino acids by potassium permanganate. Subsequent to the findings by ultraviolet difference spectroscopy that chlorine readily attacks tryptophan (Chapter IV), it was decided to examine the possibility that preferential attack of tryptophan by chlorine occurs in the presence of cystine. The oxidation of tryptophan by chlorine - 77 -

(Pigure 3.7) was found to produce a wave of half-wave potential of approximately -1.23 volts. From the results of the polarographic analyses of the oxidation of a mixture of cystine and tryptophan by chlorine (shown in Figure 3.8) it is clea~ that concomitantly with a decrease in height of the cystine wave there is an increase in the wave at -1.23 volts. It is reasonable, therefore, to conclude that chlorine oxidizes cystine and tryptophan simultaneously. (iii) Oxidation by potassium permanganate in the presence of sodium chloride. The potentiometric investigation presented in Chapter II demonstrated that the presence of large concentrations of sodium chloride increased the rate of oxidation of cystine by permanganate but there was insufficient data to establish the actual function of the salt. The fact that chlorine also reacts rapidly with cystine suggested that the oxidation in the presence of salt by permanganate may proceed by a chlorination reaction, resulting from a primary oxidation of chloride ions to chlorine. To investigate this possibility, the oxidation by permanganate of cystine alone, and together with tyrosine, in the presence of salt was undertaken and the results of this investigation are shown in Figures 3.9 and 3.10. The experimental procedure required some modification since large concentrations of chloride ions were found to FIG. 3. 7 - Polarographic wave produced by the oxidation of tryptophan by 0. 25 ml. of 0. lN chlorine.

Curve from O volts. 200 m/v abscissa unit. FIG. 3. 8 - Oxidation by chlorine of a mixture of cystine and tryptophan.

Curves: (i) 0.00ml.; (ii)0.25ml.; (iii) 0.S0ml.; (iv)0.75ml.; (v) 1. 00 ml., 0. lN chlorine. Curves (i) - (v) from O volts. 200 mv/abscissa unit. FIG. 3. 9 - Oxidation of cystine in concentrated sodium chloride solution by potassium permanganate.

Curves: (i) 0.00ml.;(ii)0.25ml.; (iii) 0.50ml.; ~) 0.75ml.; (v) 1. 00 ml., 0. lN KMnO4. Curves (i) - (v) from O volts. ZOO mv/abscissa unit. FIG. 3. 10 - Oxidation of a mixture of cystine and tyrosine in concentrated sodium chloride solution by potassium permanganate.

Curves: (i) 0. 00 ml.; (ii) 0. 25 ml.; (iii) 0. 50 ml.; (iv) 0. 75 ml.; (v) 1. 00 ml. , 0. 1 N KMn0 4 . Curves (i) - (v) from O volts. ZOO mv/abscissa unit. - 78 - affect the polarographic wave of cystine. This was overcome by employing more concentrated solutions of the amino acids (0.25 ml. of 0.05M amino acid) in brine and of the oxidant (lN) and these were allowed to interact before dilution to volume in a 25 ml. volumetric flask, resulting in a marked dilution of the salt. It is evident from these results that permanganate readily oxidizes cystine alone in the presence of salt but has very little effect on cystine when tyrosine is present in addition to the salt. The latter result is similar to that observed for the mixture when salt is excluded but differs entirely from that occurring during the chlorination of the mixture of cystine and tyrosine. Therefore, it may be concluded that the oxidation of amino acids in salt solution by permanganate occurs by the permanganate oxidizing the amino acid and not the salt and therefore, the reaction is not one of chlorination. (iv) The polarography of partial oxidation products of cystine. The polarographic behaviour of a number of the partial oxidation products of cystine has been determined117 and these are presented in Table 3.1. - 79 -

TABLE 3.1 The Polarography of Some Derivatives of Cystine. All values were measured versus a saturated calomel electrode at 2000 at a dropping mercury electrode. Half-wave potential (!0.02V) Compound in O.lN H2S04 Cy.S.S.Cy -0.86 Cy.S.SO.Cy -o.32v, -o.aav Cy.S.802.CY -0.36V Cyetine monoxide appears to yield two polarographic waves which could possibly be explained by the disproportionation,

2 Cy.S.80.CY--➔ Cy.s.s02.CY + Cy.S.S.Cy however, it is doubtful whether the conditions employed would have been severe enough to cause such a disproportionationll7. A:n interesting feature, connnon to the polarograms of cystine oxidized by permanganate or chlorine, is the appearance of a wave at a half-wave potential of -0.34V, in addition to the cystine wave occurring at a half-wave potential of -0.85V. This wave is clearly seen in the polarograms of cystine oxidized by permanganate and is only just discernible in the polarograms of cystine oxidized by chlorine. The chemical species giving rise to this second wave has not been elucidated but, since it is also present in oxidations by chlorine, it is clear that it is not associated with any manganese salt. Further, this second wave is not caused by cysteic acid since this compound is not reduced polarographically at the dropping - 80 -

mercury electrode in the voltage range employed. The fact that this wave does not increase progressively as the oxidation proceeds implies that it is a partial oxidation product of cystine which is ultimately oxidized to cysteic acid. The half-wave potential of -0.34V for this species appears to lie between that of cystine monoxide and cystine dioxide, but since the polarographic behaviour of the monoxide has not been definitely established the assignment of this wave to a partial oxidation state of cystine must be postponed.

(v) A polarographic examination of the oxidation of cysteine by potassium permanganate.

The results obtained by the potentiometric titration of cysteine with acidified potassium permanganate indicated that the oxidation of the thiol does not proceed through the formation of a disulphide bond. It was decided to obtain further information concerning this oxidation by the alternative technique of polarography.

Cysteine yields an anodic wave (half-wave potential -0.37V) due to its reaction with the dropping mercury electrodell5 and this may be represented by the following equation,

Cy. S. H + Hg~ Cy.S.Hg + W + E The polarographic waves due to cysteine and cystine present tot;ether in l;~ sulphuric acid at co11centrations of 5 x 10-4M and 4 2.5 x 10- M respectively are shown in Figure 3.11. If' -4 FIG. 3. 11 - The polarographic waves of (i) cysteine, 5 x 10 m; -4 (ii) cystine, Z. 5 x 10 m.

0

ZOO mv/abscissa unit - 81 - the oxidation of cysteine does produce the disulphide then as the oxidation proceeds the wave produced by the reduction of the cystine at -0.85V should increase in size. Figure 3.12 shows the polarographic analyses of the oxidation of cysteine by permanganate in which there is little or no sign of the cystine wave. This implies that the oxidation of the cysteine proceeds directly to the sulphinic or sulphonic acid stage and not to the disulphide. (vi) The spatial conf'iguration of the disulphide bond in relation to its reactivity in proteins. It is a well established fact that the disulphide bonds of combined cystyl residues in keratin differ in reactivity, for example, only 30% of the combined cystine in wool may be oxidized by acid permanganate. Several explanations for this behaviour have been proposed, such as the nature of the adjacent amino acid residues or the morphology of the fibre but none have received wide acceptance. It is thought that a possible explanation may lie in the steric arrangement of the disulphide bonds in proteins. The bond angles of divalent sulphur may be attributed to various types of bondingll8: (i) pure p bonds; (ii) sp3 hybrid bonds, or (111) bonds containing d character. Paulins119 considers that sulphur bonds are almost pure pin character with about 1% a-bond contribution. This would FIG. 3. 12 - Potassium permanganate oxidation of cysteine

Curves: (i) 0. 00 ml.; (ii) 0. 125 ml.; (iii) 0. 25 ml.; (iv) 0. 375 ml.; (v) 0. 500 ml., 0. lN KMn04 . Curves (i) - (v) from 200 mv. 200 mv/abscissa unit. - 82 - predict an optimum bond angle of 105° which is what is actually observed. The two remaining non-bonding electron psi.re are assigned to the a orbital and to the one remaining p orbital. The dihedral angle of 90°, found in simple alkyl disulphides, is thought to arise from repulsion between the non-bonding p orbitals on adjacent sulphur atoms. In a ring compound the dihedral angle is fixed by the bond angles. If a bond angle of 105° is assumed, the dihedral angles for a ring of 6, a, 10 and 12 sulphur atoms are 69, 103, 119 and 129° respectively. sp3 orbitals may be responsible for the bond angles in divalent sulphur compounds, since these angles correspond to the tetrahedral angle of 109°. Therefore, if sulphur bonds through an sp3 hybridization mechanism, the 90 to 110° optimum dihedral angle may be explained as arising from the repulsion of the non-bonding electron pairs. The use of d orbitals to explain the bonding has also been proposed120• It can be shown that bond angles near 110° may be constructed using 26% s bond character and anything from Oto 75% d orbitals, the remainder being p orbital. Another point of direct interest concerning the spatial configuration of the disulphide bond in proteins is the barrier to rotation of the S-S bond. For methyl disulphide the barrier for rotation about this bond has been calculated to be 6.8 k.ca1121• In cyclic compounds containing disulphide bonds, the optimum dihedral angle cannot be satisfied without - 83 - distortion of the valence angles. The disulphide bond in the ring of 1,2-dithiolane-4-carboxylic acid has a dihedral angle of 26.60 122, and that of l,2-dithione-3,6-dicarboxylic acid has a dihedral angle of 60° 123• Neither of these rings are planar ..

H c5 l,2-dithiolane-4-carboxylic l,2-dithione-3,6-dicarboxylic acid acid In thiuret hydroiodide, the ring is planar and it is thought that resonance stabilization of the planar ring system may provide the energy for reduction of the normal dihedral anglel24• Trithiones are also planar and also may be stabilized by resonance. NH2f'r=NH;I- S--S Thiuret hydroiodide Trithione Prom the above discussion, it is clear that a number of steric configurations exist for compounds containing the disulphide bond. For simple disulphides the most probable structure is the two-dimensional skew one (I) shown below. For more complicated molecules, including cyclic compounds, a number of steric positions are possible where the dihedral - 84 -

angle may vary from Oto 180° and these are represented by the cis structure (II) and by the trans structure (III) respectively.

I -r ~ 0~(o I II III It appears from the preceding investigation that the oxidation of free cystine by acid permanganate proceeds through the formation of a manganese (III) complex. The identity of this complex has not been elucidated but it appears that the intact disulphide bond is necessary for its formation since it is not observed initially with cysteine. Since under certain conditions potassium permanganate is known to oxidize the ethylene double bond by forming a cyclic complex125, it is possible that a similar mechanism is operating in the case of cystine. In the case of ethylene, the electrons involved in the oxidation are the two 2pz electrons and the dihedral angle between the 2p1 bond is oo. If the asswnption concerning the oxidation of cystine with permanganate is correct, it is necessary for the dihedral Ellgle of the

disulphide to rotate to o0 or 180° in order that the two 3px lone-pairs of electrons are aligned parallel to one another. In the case of free cystine this rotation can probably occur - 85 - but for combined cystine, the disulphide bond is unlikely to be free to rotate because of its very firm incorporation between adjacent polypeptide chains. As a result it is possible that only 30% of the combined cystine in wool may posses~ the required configuration for complexing with permanganate and this, therefore, may be the reason for the fact that only 30% of the cystyl residues in wool can react with acidified potassium permanganate. (vii) Oxidation by sodium persulphate. A polarographic examination of the oxidation of cystine by sodium persulphate was undertaken and the polarograrns obtained are shown in Figure 3.13. The oxidations were carried out by treating the solutions at 70°0 for ten minutes after which they were cooled, made up to volume and examined by the normal procedure. The inclusion of persulphate necessitates the use of the compensating current in order to counterbalance the wave produced by unreacted persulphate ion. The resulting polarograms are characterized by a maximum occurring at -l.13V and by two small waves possessing half-wave potentials of approximately -0.19V and -041V. Attempts to suppress the maxinrum by addition of more gelatin were unsuccessful and therefore, it is not possible to obtain a clear indication of the effect of persulphate oxidation on the polarographic behaviour of cystine. FIG. 3. 13 - Sodium persulphate oxidation of cystine

Curves: (i) 0. 00 ml.; (ii) 0. 25 ml.; (iii) 0. 50 ml., 0. lN Na2S2Os. Curves (i) - (iii) from O volts. 200 mv abscissa unit.

_, FIG. 3. 14 - Peracetic acid oxidation of cystine

Curves: (i) 0. 00 ml.; (ii) 0. 25 ml.; (iii) 0. 50 ml.; (iv) 0. 75 ml., 0. 0lN CH3CO3H. Curves (i) - 6-v) from O volts. 200 mv/ abscissa unit. - 86 -

(viii) Oxidation by peracetic acid. The effect of peracetic acid oxidation on the polaro­ graphic behaviour of cystine may be noted in Figure 3.14. It is evident that peracetic acid causes no reduction in size of the cystine wave but it does alter the shape of the wave. Thus, it is not possible to employ the polarographic procedure to investigate the oxidation of cystine by peracetic acid.

Conclusions

The above polarographic investigation has yielded evidence supporting earlier claims that potassium permanganate and chlorine react preferentially with tyrosine or cystine respectively from mixtures of these amino acids. However, since the polarographic method is applicable only to the free amino acids, it is not possible to ascertain whether this preferential attack may also occur for these amino acids combined in proteins. In order to investigate this problem it was decided to employ the technique of ultraviolet difference spectroscopy and this investigation is presented in the following chapter. - 87 -

Chapter IV: THE OSB OP ULTRAVIOLBT SPECTROSCOPY TO STUDY THE BPFECTS OF OXIDIZING AGERTS ON AMINO ACIDS A1fD PROTEINS

Introduction

ot the twenty or so amino acid constituents of proteins, only a few absorb ultraviolet radiation in the (240-340) mµ. region. These are the aromatic amino acids tyrosine, tryptophan and phenylalanine and the sulphur-containing amino acid cystine, the respective chromophores being the phenol, indole and phenyl rings and the disulphide linkage. The tyrosine and tryptophan chromophores exhibit maxima in the (270-290) mJJ. region and these are characterized by large molecular extinction coefficients. The spectrum of phenylalanine displays fine structure in the (240-275) mJJ. region but, because of its extremely low molecular extinction coefficient, its absorptivity is very low in comparison to that of tyrosine and tryptophan. The spectrum of cystine, though not characterized by a distinctive band, exhibits a gradual increase in absorptivity from 310 mJL to 250 mJJ. but, as a consequence of its small molecular extinction coefficient, large concentrations of cystine are required to produce significant absorption. 'l'he absorption characteristics of these amino acids are presented in Table 4.1. Figure 4.1 exhibits the spectra of tyrosine, tryptophan, phenylalanine and cystine, all at neutral pH, plotted as their occurrence FIG. 4. 1 - Ultraviolet absorption spectra of cystine, tyrosine, tryptophan and phenylalanine.

0.8

i::: o. 6 ....0 ....p.. f.l 0 ] o. 4 (e) <

0.2

0 250 300 350 400 Wavelength (m/"' )

(a) Tyrosine.

(b) Tryptophan.

( C) Cystine.

(d) Phenylalanine.

(e) Sum of these four amino acids . - 88 - in keratin. TABLB 4.1 Molecular Extinction Coetticients (E) ot Amino Acids in Acid Yedial26

Canpound. Amax (mJ.l) t X 10-3 tnoaine 274.6 1.34 tryptophan 287.6 4.66

278.6 6. 66 phenylalanine 260 0.144 cystine 300 0.03 290 0.06 270 0.19

264 o. 34

Only the most highly absorptive maxinnun exhibited by phenyl­ alanine is shown in Table 4.1, while the_ cystine values do not correspond to maxima in the wavelength region shown. The ultraviolet absorption spectrum ot proteins and peptides can be subdivided into two main absorbing spectral regions. The tirst region, (180-260) mJJ., is characteristic ot the peptide group (-CO.NH-) and is consequently present in both classes ot compounds. The second ultraviolet absorption region, (250-400) mJL , is dependent on the additional chromophores associated with the side chains ot the protein or peptide. Thus the characteristic absorption maxima ot - 89 - proteins in the region above 240 mJLare due primarily to their content ot tyrosine, tryptophan, phenylalanine and cystine and therefore, is the region employed exclusively in this investi- gation. The absorption spectra of the tree and combined amino acids -are not usually identical due to the tact that peptide bond foi,nation eliminates the amino and carboxylic groups' function except tor the terminal amino and carbo:xylic groups. Thus incorporation of the chromophoric amino acids into peptides has been tound to produce a loss in tine structure ot the spectra and a shift of the absorption to longer wavelengths. The magnitudes of the wavelength shifts due to the peptide formation are of the order 0.4 m.J.l for the tyrosine maxima and 0.9 mµ.. tor the tryptophan fine structure band. In proteins, however, wavelength shifts of the order

of 3 D1}l have been observed so that other factors must be operating and these have recently been reviewed by Bevan and Bolidayl27, and Wetlauter128• Despite these discrepancies, the ultraviolet absorption spectra ot proteins resemble very closely the spectra of their composite amino acids, in particular that of the major absorbing constituents, tyrosine and tryptophan, conse1,11ently, this has been made the basis of a spectrophotometric method tor estimating tyrosine and tryptophan in proteins127• It is evident from the preceding discussion that, apart trom phenylalanine which is relatively inert, the ultraviolet - 90 - absorbing amino acids tyrosine, tryptophan aDd cystine are just those which appear to be most prone to oxidation and, therefore, ultraviolet absorption spectroscopy affords a convenient method tor the study of the oxidation of proteins. However, initial experiments employing direct ultraviolet absorption analyses of oxidized proteins proved to be extremely difficult to interpret due to the fairly high protein background absorption and scattering, masking the small oxidation-induced spectral changes. To overcome this difficulty difference spectroscopy, a technique where small spectral perturbations can be separated from highly absorptive backgrounds, was employed tor this investigation. This technique has previously been success:fully applied to studies of the spectral changes occurring in the amino acids and proteins following a variety of solution changes such as pH, temperature and solventl2S as well ea protein spectral changes produced by ultraviolet 1rradiation129•

Difference Spectrophotometry A difference spectrum is usually formed by variations in spectral absorption between a control spectrum and a sample spectrum and is most conveniently obtained by means of a double beam spectrophotometer. A difference spectrum can occur in either of two ways: (1) by a perturbation of a chromophore resulting in a wave­ length shift and/or an intensification of an absorption - 91 -

band or, (11) by modification of the absorbing chranophore resulting in the formation of a new species. The difference spectrum formed by wavelength shifts of the protein.absorption is usually caused by a variation in the enviroDDent of the chromophore such as changes in pH, temperature and solvent and it may be shown that the differ­ ence spectrum in this case will have the form of the negative ot the first derivative of the original absorption curve1~0. Therefore, in any investigation of the spectral changes formed by chemical modifications of chromophores, it is necessary to ensure that spectral perturbations, produced by variations in the enviromnent of the chromophores are kept to a minimum. The difference spectrum produced by chemical modification of the chromophore is usually the combination of two effects; firstly, a negative absorption produced by the destruction of some of the original chromophore and secondly, the spectral absorption of the newly formed chemical species. If a non- absorbing chemical species is formed, the difference spectrum ia the mirror image, on the negative side of the wavelength axis, ot the original absorption spectrum. It is more likely, however, that the modification would result in the formation of a new chromophore so that the resulting difference spectrum would possess features only slightly resembling the - 92 - negative of the original spectrum. A detailed study of the difference spectral changes produced by the treatment of soluble proteins by oxidants would, therefore, yield useful information about which amino acid residues are attacked and possible oxidation products. However, in order to interpret these difference spectra it was necessary initially, to determine and characterize the spectral changes produced by the oxidation of the free amino acids tyrosine, tryptophan am cystine.

Experimental procedure In preparing standard solutions of the amino acids for ultraviolet difference spectroscopy certain precautions concerning the choice of solvent and the concentration of amino acid are necessary. Care must be taken in choosing a suitable solvent and in this regard it is necessary to consider factors such as the solubility characteristics of the amino acids and the necessity that the solutions be buffered to prevent any significant change in the pH of the system. Jurthermore the solvent and buffer employed must not absorb strongly in the ultraviolet region of interest. The solvent system employed in this investigation was a modified Universal butter comprising of acetic, phosphoric and boric acids which gave a buffered solution of pH2. This solution was found to be suitable for dissolving the small amounts of tyrosine and - 98 - tryptophan employed but to dissolve the larger amounts of cystine (necessary because of its very low molecular extinction coefficient in this wavelength region) it was necessary to reduce the solution pH to a value of 1.1 by the addition of sulphuric acid. The amino acid concentrations employed must not be as high as to render the spectrophotometer detecting system ineffective. In this regard it was necessary to employ concentrations of the amino acids which yielded optical densities not higher than 1.5 in the (240-340) 111.J.l- region. The amino acid concentrations were as follows: tyrosine 0.00075M tryptophan 0.00025M cystine 0.003M The oxidation ot these amino acid solutions was carried out as follows: 5 ml. aliquots of the amino acid solutions were pipetted into a series of test-tubes, to the sample solutions was added the required volume ot oxidant and to the reference solutions an equivalent volume of the buffer solution was added. In cases where high temperatures were required tor the oxidative treatments, it was necessary after the treatment to dilute both reference and sample solutions to 10 ml. in a volumetric flask in order to compensate tor evaporation. On ccmpletion of the oxidation, the difference spectra produced were recorded on either a Beckman D.K.2 or - 94 - a Cary model 15 recording spectrophotometer.

The Oxidation of the Amino Acids 1. The oxidation of tryptophan. The difference spectra, produced by the oxidation of tryptophan by potassium permanganate, potassium permanganate/ sodium chloride, chlorine, sodium persulphate and peracetic acid, were found to possess the same basic features (see Figures 4.2 to 4.13). These spectra are characterized by positive optical densities above and below 260 and 295 m_µ. respectively and by negative optical densities between these two wavelengths. The latter region of the difference spectra resembles closely the mirror image, on the negative side of the zero optical density line, of the normal absorption spectrum of tryptophan, even to the detail of fine spectral features which occur at almost identical wavelengths to their counterparts in the tryptophan absorption spectrum. It is reasonable to conclude, therefore, that this feature of the difference spectra is an abstraction spectrum resulting from the disappearance of some of the original tryptophan in the sample solution by oxidation. The positive optical densities above 295 m must arise from the oxidation products of tryptophan, since neither tryptophan itself nor the reduced form of the oxidants absorb at these wavelengths. These two spectral features, therefore, may be utilized FIG. 4. 2 - Ultraviolet difference spectra. Permanganate oxidation of tr 3/4 hours at room temperature). 0.3 d

0.2

o. 1 e d >- 0 ....+> C ID b ~ -0.1 "Cl a -....~ -0.2 +> b' -0. 3

-0.

-0.5.__...... ,..,,.... _ ___...___ ...... _ ___. _ _.,_.....____...__.___.,_ ...... _ 240 260 280 300 320 340 Wavelength (m)"-) Curves: (a) 10.,u.l.; (b) 20.,.u.1.; (c) 30 ,,µ.1.; (d) 40_µ...1.; (e) 50)-"'-1., of 0. lN KMnO4

FIG. 4. 3 - Ultraviolet difference spectra. Permanganate oxidation of tryptophan in sodium chloride (1 3/4 hours at room temperature).

0.3

0.2

0. 1 e >- ....+> d ID 0 r:: C (I) "Cl b .-I -0.1 «I a ....u +> -0.2 p.. 0 -0.3

-0.5

240 260 280 300 320 340 Wavelength

Curves: as above FIG. 4. 4 - Ultraviolet difference spectra formed by oxidation and tryptophan with chlorine for 30 minutes at room temperature.

0.3

0.2 >- ...... 0. 1 U) ~ C Q) 'O 0 b r-4 «I a ....u -0. ...p,. 0 -0.2

-0.3

240 260 280 300 320 340 Wavelength (m,.,.u-) Curves: (a) 10 p-1.; (b) 20 JA-1.; (c) 30 r,l., of 0. lN chlorine.

FIG. 4. 5 - Ultraviolet difference spectra formed by oxidation of tryptophan with sodium persulphate for 15 hours at room temperature.

a fi 0.2 a ...... >- ID ~ 0. 1 a Q) fi ,t, a r-4 0 «I ....u ...p,. -0.1 0 -0.2 g ae -0.3

240 2 0 280 300 320 340 Wavelength (m,)-"--)

Curves: (a) 10.)A-l.; (b) 20 p-1.; (c) 30.JA-l.; (d) 40 _,,.u-1. ; (e) 50j-tl., of 0. lN sodium per sulphate. FIG. 4. 6 - Ultraviolet difference spectra formed by per sulphate oxidation of tryptophan for 10 minutes at 606 c.

e 0.3

0.2 ...... >- ID d 0.1 Q) "C e .-4 0 d RI CJ C ...... -0.l p.. b 0 a -0.2

C -0.3 d e 240 2 0 280 300 320 340 Wavelength (Irlj-1-) Curves: (a) 2014 l. ; (b) 40 ;d. ; (c) 60 _,M,l. ; (d) 80 fol. ; (e) 100_µ.1. , of 0. lN sodium persulphate. FIG. 4. 7 - Ultraviolet difference spectra formed by persulphate oxidation of tryptophan for 10 minutes at 70°c. o.s

0.4

0.3

0.2 ...... >- !I.I d 0.1 Q) "C s 0 a RI -....CJ p,. -0.1 0 -0.2

-0.3

240 2 0 280 300 320 340 Wavelength (m_,,A-L ) Curves: (a) 20fol.; (b) 40_.µ.l.; (c) 60J-A-l., of 0. lN in sodium per sulphate. FIG. 4. 8 - Ultraviolet difference spectra formed by persulphate oxidation of tryPtophan for 10 mine. at 80°c. d e 0.5

0.4 C 0.3 >, •M.... CD 0.2 e s::: Q) d o. 1 0 C .-1 Ill u 0 b •M.... a p,. a 0 -0.1 b ae -0.2

-0.3

240 260 300 320 340 Wavelength (mp.) Curves: (a) 20_,A..t.1.; (b) 40 pl.; (c) 60_µ.1.; (d) 8~1.; (e) 1ogu..1., of 0. lN sodium persulphate.

II FIG. 4. 9 - Ultraviolet difference spectra formed by per sulphate

oxidation of tr to han for 10 mins. at 89°c. II 11" 0.5 :I 0.4 I

....>- a •M 0.3 CD s::: Q) 0.2 'tj .-1 Ill u 0.1 •M.... 8' 0

-0.1

-0.2

260 280 300 320 340 Wavelength (m)-"-)

Curves: (a) 20)-'-l.; (b) 40)-'--1.; (c) 60..,.u-1., of 0. lN sodium per sulphate. FIGS. 4. 10 , 4. 11 , 4. 12 - Ultraviolet difference spectra of tryptophan oxidized by peracetic acid for 10 mins. at SO, 70 and 82. S0 c respectively.

0.2 4.10

>-+0.2 ....+> en 4. 11 r::= .,,Q) g 0 ~------=:S~-----:::--;~::::S~-...:a

4.12 0. 1 g o~______:~~,,-----___,,~~!!!!!...-a -0.1

240 260 280 300 320 340 Wavelength (l'lljA-).

Curves: (a) 20,µ.1.; (b) 40 JA,L; (c) 60.fLl.; of O. IN peracetic acid. FIG. 4. 13 - Ultraviolet difference spectra of tryptophan oxidized by permonosulphuric acid for 10 minutes.

0.4 >- ...... 0.3 II) d Q) 'tj 0.2 .-I nl ....u 0. 1 ..p. a b 0 0

-0. 1

240 260 280 300 320 340

Wavelength (IlljL )

0 0 Curves: (a) 60_,,u..l.; 50 c; (b) 60.,,u-l., 80 c., of 0. IN permonosulphuric acid. - 96 - in studying the effects ot various oxidizing agents on tryptophan. (1) OXidation by potassium permanganate. The difference spectra produced by the oxidation ot tryptophan by·potassium permanganate are shown in Pigure 4.2. 'l'bese spectra are characterized by positive optical densities above 296 mµ and below 260 mJ.l and by the minima, ot negative optical densities, occurring at 280 and 288 mp.. The negative canponent of the difference spectrum resembles closely the shape ot the normal absorption spectrum ot tryptophan, tor example, the difference minima at 280 and 288 mJJ.. correspond closely to the absorption maxima at 278.5 and 287.5 m).l respectively and, furthermore, the difference spectra possess a shoulder at 273.6 mJl corresponding to one at 272.5 mJ.l. ot the tryptophan absorption spectrum. It 1s likely, therefore, that the difference spectra arise from the formation ot new ultraviolet absorbing chromophores, resulting in the observed absorption at wave­ lengths above 300 m.JJ. and the disappearance of some of the original tryptophan in the oxidized sample as evidenced by the difference spectral minima at 280 and 288 mJ.l. The tact that this series ot difference spectra possesses the same structure throughout signifies that the oxidation is proceeding by the same mechanimn. Thus, in this case, the magnitudes ot the optical densities at 280 and 306 mJJ. give an indication of - 96 - the amount of tryptophan oxidized and the amount ot the new chranophore formed respectively. Clearly, as more oxidant is added to the tryptophan solution there is a corresponding change in the optical densities at these two wavelengths, designating an increase in the amount of tryptophan oxidized and the new chromophore produced. An interesting feature of this series of difference spectra is the appearance of two isosbestic points of zero optical density at 262.5 and 294.5 mJ-l• These points, however, do not correspond to iaosbestic behaviour in normal absorption spectroscopy. The occurrence of isosbestic points in the difference spectra of oxidized tryptophan indicates that, at these wavelengths, the sum- of absorbances of the oxidized and residual tryptophan remains constant during the oxidation reaction and consequently, this implies quantitative conversion of tryptophan to its reaction products. This behaviour may be tested by plotting the optical densities at 280 and 306 mJJ- against one another for each spectrum of this series and this is shown in Figure 4.14; the linearity of this plot testifies to the validity of the quantitative oxidation of tryptophan. The fact that the isosbestic points occur at zero optical density indicates that the sum of the absorptivities of oxidized and residual tryptophan must equal the absorptivity ot umnodified tryptophan in the reference cell and this infers FIG. 4. 14 - Plot of optical densities (negative) at 280 m,,u. versus optical densities (positive) at 306 mµ. for Fig. 4. 2.

-0.S

} 0 co N -0.4

...Cl! ...... >- en i:: Q) 'O -0.3 Cl! -....u ...p,. 0

-0.2

-0. I

0 L------'------'----- 0. l 0.2 0.3

Optical density at 306 o/ - 97 - that the mixture of the oxidation products of tryptophan has the same molecular extinction coefficient as tryptophan itself at these two wavelengths. The difference spectra formed by the oxidation ot tryptophan by. potassium permanganate in a concentrated solution of sodium chloride are shown in Figure 4.3. Clearly, these spectra are virtually identical to those formed when salt is excluded trom the system indicating that the presence of sodium chloride has little effect on the oxidation of this amino acid by potassium permanganate. (11) Oxidation by chlorine. The difference spectra arising from the oxidation of tryptophan solution by chlorine are shown in Figure 4.4. These spectra closely resemble those produced by the perman­ ganate oxidation of tryptophan and therefore, the spectral features may be attributed to similar factors as enunciated earlier. This would indicate that permanganate and chlorine oxidize tryptophan in much the same way. (111) Oxidation by sodium persulphate. The oxidation of tryptophan by sodium persulphate appears to be extremely dependant on the temperature. At room temperature the oxidation is very slow as evidenced by the low absorptivities, both negative and positive, of the difference spectra generated after a reaction time of 15 hours (Pigure 4.5). Above 50°0, the oxidation proceeds more - 98 - rapidly as can be seen from the swift production ot the difference spectra, requiring reaction times of only ten minutes (Pigures 4.6 to 4.9). 'l'hese results are consistent with the known kinetics of persulphate i~n oxidations131 which involve either the bimolecular reaction between the reductant (R) and persulphate,

(1) or by the thermal decomposition of the persulphate ion,

(2)

At low temperatures, the rate of thermal decomposition of persulphate ion is low and the oxidation probably proceeds by the bimolecular reaction (1). At higher temperatures, the rate of thermal decomposition of persulphate ion becomes comparable and eventually exceeds reaction (1) and the oxidation proceeds mainly by the free radical mechanism (2). The difference spectra of persulphate oxidized tryptophan possess the same overall structure as described earlier for trYPtophan oxidized by permanganate and by chlorine. It is reasonable to conclude, therefore, that the negative ccmponent of the difference spectra results from the destruction of some of the original tryptophan, whereas the poaitiTe absorptions are caused by the new chromophores arising from the oxidation ot tryptophan. - 99 -

At temperatures above 70°0 the difference spectra, illustrated in Pigures 4.8 to 4.9, show extremely high positive optical densities, characterized by a distinct maximum at approximately 302 m_µ, which are not evident in the difference spectra generated at lower temperatures. This large increase in positive optical densities is not accompanied by a concurrent increase in the negative optical densities at 278.5 and 288 111),t. and from this it would appear that there is little additional loss in tryptophan at these elevated temperatures. It appears, therefore, that the maximum occurring at 302 m_µ. is caused by additional chromophores, resulting from the attack of peraulphate on the original oxidation-induced chromophores. The lack of isosbestic behaviour in these aeries of difference spectra indicates that the attack of tryptophan by peraulphate is more complex than with the other oxidants. (iv) Oxidation by peracetic acid. The oxidation of tryptophan by peracetic acid at roan temperature was found to be extremely slow and therefore, the oxidation was carried out at elevated temperatures. The difference spectra produced by this oxidation are shown in Pigurea 4.10 to 4.12 from which it is evident that the overall ehape of these spectra is similar to that induced in tryptophan

by the oxidants discussed earlier. It is of interest to note that the positive optical - 100 - densities above 297. 6 mJ.L are wilike those formed by the other oxidants already examined, for in this case the absorptions cut out at 320 mJ.l whereas those already investigated extend beyond 340 mJJ.. This observation indicates that peracetic acid oxidizes tryptophan to a different chromophore than is formed by the other oxidants. (v) Oxidation by permonosulphuric acid. Difference spectra of very low absorptivities (shown in Pigure 4.13) were generated by treating tryptophan with permonosulphuric acid at elevated temperatures. This behaviour suggests that either permonosulphuric acid reacts very slowly with tryptophan under these conditions or else the oxidation is localized on the alkyl side chain of this amino acid, since this would result in only minor perturbations ot the chromophoric indole ring. (vi) Interpretation of spectr~l changes. The difference spectra generated by the oxidation of tryptophan are characterized by an "abstraction" pattern, arising from a loss of part of the original tryptophan in the oxidized sample. The "abstraction" characteristics are evident from the similarity of shape of the difference spectrum with the normal absorption spectrum of tryptophan as shown in Pigure 4.15. The remaining components of the difference spectrum undoubtedly arise from the oxidation products of trn,tophan. FIG. 4. 15 - Ultraviolet spectra of tryptophan.

0.9

0.8

0.7 0.6

0.5

>, 0.4 ....+-' Ill (a) i::: Q) 0.3 'O ~ro 0.2 ....u +-'p.. 0. 1 0 0

-0.2 (b) -0.3

240 260 280 300 320 340

Wavelength (mf-L)

(a) Absorption spectrum. (b) Difference spectrum resulting from oxidation. - 101 -

The formation ot the "abstraction" feature, of negative optical density, is thought to arise from the disruption ot the indole ring, resulting in the formation ot various products such as,

kynurenine,

~COOH anthranilic acid, V-NH~ kynurenic acid, c&co~ 3-hydroxyanthranilic acid, ~COOHVNH~ OH The ultraviolet absorption spectra ot these compounds, together with those ot tryptophan and indolyl-3-acetic acid, are shown in Pigure 4.16; these spectra were recorded tor 2 X 10-411 solutions ot these compounds in sulphuric acid. It is evident that the spectra of kynurenine, kynurenic acid, anthranilic acid and 3-hydroxyanthranilic acid possess lower molecular extinction coefficients than tryptophan in the (267-293) mJ.L region. Thus, the oxidation ot tryptophan to products such as these would yield, in this wavelength region, a component ot negative optical density in the ditference spectra of oxidized tryptophan and this is actually observed experimentally. FIG. 4-16 - Ultraviolet absorption spectra of tryptophan and some possible oxidation products of tryptophan.

All solutions 0. 0002M in sulphuric acid.

0.9

0.8

>- 0.7 ....+> rn s:: 0.6 Q) re, .-4 «I 0.5 u ....+> p,. 0.4 0 0.3

0.2

0. 1

0 220 240 320 340 380

Wavelength (mj-4- )

(a) Kynurenine. (b) Anthranilic acid. (c) 3- hydroxy anthranilic acid. (d) Kynurenic acid. (e) Tryptophan. (f) Indolyl-3-acetic acid. - 102 -

This view 1s supported by the fact that the absorption spectra ot indolyl-3-acetic acid and tryptophan are virtually identical and it would appear, therefore, that oxidative modification of the alkyl side chain of tryptophan has little effect on the absorption characteristic of the indole nucleus and consequently, could not give rise to the difference spectral component of negative optical densities in the wave­ length region (270-294) mµ. The striking similarity in the difference spectra of tryptophan oxidized by a variety of oxidizing agents suggests that these oxidations proceed to the formation of similar products irrespective of the particular oxidant. Thie suggestion was examl.ned in the following manner: Concentrated solutions of tryptophan in dilute sulphuric acid were oxidized with potassium permanganate, peracetic and permonosulphuric acids. In the case of the peracids it was necessary to wait several days for the oxidation to proceed to a detectable level. The oxidized solutions were diluted with methanol and then spotted on a thin layer chromatographic plate coated with silica gel. The chromatographic separation was then carried out in a glass cabinet employing butanol, acetic acid, water (120:80:50, respectively) as solvent. The plates were then removed and dried in an air-draught oven for 16 minutes at 105°c when they were allowed to cool and sub­ sequently examined in the following sequence: - 103 -

(1) Fluorescence under ultraviolet irradiation with a Phillips high-pressure mercury discharge lamp. The fluorescent products are shown in Figure 4.17. (11) Colour develo1111ent with ninhydrin (Figure 4.18). (111) Colour development with Erlich's reagent (Figure 4.19). (iv) Colour development with sulphanilic acid reagent (Pigure 4.20). Prom the large number of oxidized derivatives formed, it is clearly not possible to identify everyone of these. However, there is sane evidence to show that kynurenine is readily formed in the case or permonosulphuric acid oxidation or tryptophan, but less readily with permanganate and peracetic acid oxidations. The lack of purple colours after treatment of the chromatograms with Erlich's reagent point to the fact that the indole ring has been disrupted by the oxidation treatments, in accordance with the results obtained by ultra­ violet difference spectroscopy. It is clear from Figures 4.17 to 4.20 that a number or conmon products are formed by the oxidation of tryptophan by these oxidants. 2. The oxidation or tyrosine. 'l'he difference spectra produced by the oxidation of tyrosine with potassium permanganate, potassium permanganate/ aodiwn chloride, chlorine and eodiwn persulphate are shown in Pigures 4.21 to 4.24, from which it is evident that these spectra have many conmen features. FIG. 4. 17 - Fluorescence under ultraviolet irradiation of thin layer chromatograms of oxidized tryptophan.

Solvent front

0 0

0 p oO o 0 0 0 0 0 0 0 0

Start 0 0 • 0 0 0 0 A B C D A E F

A. N-methyl tryptamine, kynurenine. B. Permanganate oxidized tryptophan. c. Kynurenine. D. Peracetic acid oxidized tryptophan. E. Permonosulphuric acid oxidized tryptophan. F. Tryptophan. FIG. 4. 18 - Ninhydrin positive spots of thin layer chromatograms of oxidised tryptophan.

Solvent front

0 0 00 0 0 0 0 0

Start • • 0 0 • 0 A B C A E F

A. N-methyl tryptamine, kynurenine. B. Permanganate oxidized tryptophan. C. Kynurenine. D. Peracetic acid oxidized tryptophan. E. Permonosulphuric acid oxidized tryptophan. F. Tryptophan. FIG. 4. 19 - Thin layer chromatograms of oxidized tryptophan: positive reaction with Erlich' s reagent.

Solvent front

0 0 0

0 0 0 0 0 0

0

Start • 0 • • • 0 A B C D A -E F

A. N-methyl tryptamine, kynurenine. B. Permanganate oxidized tryptophan. C. Kynurenine. D. Peracetic acid oxidized tryptophan. E. Permonosulphuric acid oxidized tryptophan. F. Tryptophan. FIG. 4. 20 - Thin layer chromatograms of oxidized tryptophan: sulphanilic acid reaction.

Solvent front

0 0 0

0 0 0 Start 0 0 0 0 A B C D A E F

A. N-methyl tljptamine, kynurenine. B. Permanganate oxidized tryptophan. C. Kynurenine. D. Peracetic acid oxidized tryptophan. E. Permonosulphuric acid oxidized tryptophan. F. Tryptophan. - 104 -

The difference spectra arising from the oxidation of tyrosine are characterized by a maximum occurring in the vicinity of 286-289 mJJ.. and by a broad minimum between 260 and 280 mµ.. There is also a gradual increase in the optical densities below 250 D1JL • The appearance of these features may be attributed to: (1) the formation of new chemical species, resulting in a general increase in the optical densities of the oxidized sample at wavelengths between 280 and 340 mµ. , and (11) the loss of some of the original tyrosine from the oxidized sample, producing an "abstraction spectrwn" as evidenced by a decrease of optical density in the (260-280) mJ-l region. This conclusion is supported by the fact that the difference spectral minimum is similar in general shape to that of the normal tyrosine absorption spectrum, even to the detail of exhibiting a shoulder at approximately 280 mJ.l. The fact that the difference spectrum is always positive indicates that the compounds to which tyrosine is modified are more strongly absorptive in the C:?60-280) m_µ. region than is tyrosine itself.

(1) Oxidation by potassium permanganate. The difference spectra produced by the oxidation of tyrosine with potassium permanganate ere shown in Pigure 4.21. FIG. 4. 21 - Ultraviolet difference spectra formed by oxidation oft rosine with otassium erman anate for 20 mins. at room tern . 0.7 0.6

0.5

0.4 >- ...... I'll 0.3 i::: Q) "(j ..... 0.2 111 (J ..... 0. l ....p. 0 0

-0. l

240 260 280 300 320 340 Wavelength (m.,.u. ). Curves: (a) 10_,..u, 1.; (b) 20.,.u.l.; (c) 30_,u..l.; (d) 40_,µ- l.; (e) SO.fol., of 0. lN potassium permanganate. FIG. 4. 22 - Ultraviolet difference spectra formed by oxidation of tyrosine in the presence of sodium chloride with potassium perman­ anate for 20 minutes at room tern erature. e 0.4

0.3 >- ...... 0.2 I'll i::: Q) 0. 1 "(j ..... 111 ....u 0 ....p. 0 -0. 1

-0.2 240 260 280 300 320 340 Wavelength (m,,u- ).

Curves: (a) 10_,M-l.; (b) 20_,M,-l.; (c) 30.)-'-l.; (d) 50.)"-l.;ofO.lN potassium permanganate. FIG. 4. 23 - Ultraviolet difference spectra formed by oxidation of tyrosine with chlorine for 1 3/4 hours at room temperature.

0.7

0.6

0.5 ...>- ·~Ill 0.4 i::: Q) 't) 0.3 ell -(.) ·~... 0.2 0. 0 0. 1

0

-0. 1

240 260 280 300 320 340 Wavelength (m.J-L).

Curves: (a) 10 µl.; (b) 20 _µl.; (c) 30 _µI.; (d) 40_µ.l.; of 0.804% active chlorine.

8 8

......

-a -a

......

......

+> +>

~ ~

RI RI

ID ID

Q) Q)

s::: s:::

u u

0.1 0.1

0.2 0.2

0 0

FIG. FIG.

240 240

4. 4.

24 24

Curves: Curves:

-

Ultraviolet Ultraviolet

persulphate persulphate

(a) (a)

sodium sodium

260 260

40µ1.; 40µ1.;

difference difference

persulphate. persulphate.

for for

g g

e e

a a

Wavelength Wavelength

(b) (b)

10 10

80J-ll.; 80J-ll.;

minutes minutes

280 280

spectra spectra

(mp. (mp.

(c) (c)

at at

formed formed

70°c. 70°c.

). ).

120,.,u.l.; 120,.,u.l.;

b b

a a

a a

C C

300 300

by by

oxidation oxidation

(d) (d)

160~1.; 160~1.;

of of

tyrosine tyrosine

320 320

(e) (e)

200_.,u.l., 200_.,u.l.,

with with

sodium sodium

340 340

of of

0. 0. IN IN - 106 -

This aeries of difference spectra is characterized by a broad minimum between 260 and 280 m_µ.. and by a distinct maximum at 288.5 mJJ., in addition there is also a shoulder at 280 mJA,• Evidently, from Figure 4.21, as the oxidation of tyrosine proceeds there is an increase in the absorptivity of the difference spectra, particularly at the vicinity of the maximum at 288.5 mJ.L. It was thought, therefore, that the magnitude of the optical density at 288.5 mJJ. should give some indication of the extent of oxidation occurring. The oxidation of tyrosine in concentrated solutions of sodium chloride was also studied and the difference spectra generated in this case are shown in Figure 4.22. These spectra, although similar, are not identical to those induced in the absence of salt and clearly, the absorptivities at corresponding amounts of added oxidant are lower when salt is present, suggesting that the presence of sodium chloride retards the rate of oxidation of tyrosine by potassium permanganate. (11) Oxidation by chlorine. The difference spectra formed by the oxidation of tyrosine with chlorine are shown in Figure 4.23 and are very similar in shape to those produced by the action of potassium permanganate on tyrosine. It would appear, therefore, that these difference spectra result mainly frcrn the same factors already considered, namely, the generation of new chemical

species derived from tyrosine and the loss of some of the - 106 - original tyrosine in the oxidized sample. There is a slight shift in the wavelength position of the maximUJD feature towards lower wavelengths and this feature now occurs at 286.5 mµ. in comparison to 288.5 mµ. in the case of permanganate ~xidized tyrosine. This anomaly is thought to arise from the fact that the species produced by these treat­ ments are not identical and consequently would be expected to have different absorption characteristics. It is evident from the increase in magnitude of the optical density of the difference spectra at 286.5 mJJ.. that the extent of oxidation of tyrosine increases with added oxidant. (111) Oxidation by sodium persulphate. The di:fference spectra generated by the action of sodium persulphate on tyrosine at 70°0 after a treatment time of ten minutes are shown in Figure 4.24. The shape of this series of difference spectra resemblesclosely those produced by oxidizing tyrosine with permanganate and chlorine, except in this case the overall absorptivity of these spectra is much lower, even allowing for the dilution to constant volume to provide for losses due to evaporation. It would appear, therefore, that persulphate does not readily attack tyrosine. (iv) Oxidation by peracetic and permonosulphuric acids. It was found that no significant difference spectra were formed by treating tyrosine with either oxidant at temperatures varying from 20° to 70°c indicating that tyrosine is not attacked - lO'l - by these reagents under the experimental conditions employed. However, et higher temperatures and longer reaction times, such as heating over a steam bath for 24 hours, it is found that these peracids can oxidize tyrosine. The products formed by this trea~ent have been subjected to a one dimensional thin layer chromatographic separation, employing a similar procedure to that described for the tryptophan oxidation, and the chromatograms after ninhydrin develoJ;1J1ent are shown in Figure 4.25. It is evident from these separations, that a large variety of products are formed on oxidation of tyrosine but the identification of these products was outside the scope of the present investigation.

3. The oxidation of cystine. The difference spectra produced by the oxidation of cystine with potassium permanganate, potassium permanganate/ sodium chloride, chlorine, sodium persulphate, peracetic and perrnonosulphuric acids are shown in Figures 4.26 to 4.32. It is well established that these oxidants can bring about the oxidation of cystine fairly readily but the spectral characteristics of the products appear not to be sufficiently different from that of unmodified cystine to result in distinctive difference spectra. This, therefore, renders the use of difference spectroscopy unsuitable for investigating the oxidation of cystine alone or in mixtures with tyrosine or tryPtophan. FIG. 4. 25 - Thin layer chromatograms of oxidized tyrosine: positive ninhydrin reaction.

Solvent front

0 0 0

00 -- 8 0 0 .,, Start " • JI 'It • " 1' ,c A A B C C B D D B

A. Sodium persulphate oxidized tyrosine. B. Unmodified tyrosine. C. Permonosulphuric acid oxidized tyrosine. D. Per acetic acid oxidized tyrosine. FIGS. 4. 26, 4. 27 - Ultraviolet difference spectra formed by potassium permanganate oxidation of cystine in the absence and presence of sodium chloride respectively for 20 mins. at room temperature.

~ 0.2 t a 4.26 0. 1 ...... >- 0 m s::: Cl) o. 1 "d .-4 nl u 0.2 ...... p.. 4.27 0 0. 1 d

-0. 1

240 260 280 300 320 340

Wavelength (m;t-),

Curves: (a) 10),Ll.; (b) 20.fA-l.; (c) 30_µ.l.; (d) 40}'-l.; (e) 50.,u.l., 0. lN potassium permanganate. FIGS. 4. 28, 4. 29, 4. 30, 4. 32 - ultraviolet difference spectra of cystine oxidized by chlorine, sodium per sulphate, peracetic and permonosulphuric acids, respectively.

o. 1 (b) 3¼ hours at room temp. 4.28 0 >- 0. 2 ...... (b) fll ~ o. 1 4.29 a, a i:, .-4 0 nl 0.3 ...... u p,, (b) o o. 2 10mins. atS0°c. 0. 1 4.30

0 0. 1 0 (b) 10 mins at 50 c. 4.32 0

240 260 280 300 320 340 Wavelength (m,.u.. ).

Curves: (a) 40 ,,u.L ; (b) 60 ,.ul. ; oxidant. - 108 -

It is clear from the foregoing investigation that the technique of difference spectroscopy is ideally suited for investigating the oxidation of free tyrosine and tryptophan since these treatments result in the formation of character­ istic spectra. It is possible, therefore, that modification of combined tyrosine or tryptophan in proteins may also be discernible by the formation of analogous difference spectra. One factor which mu.et be considered in this connection, when comparing the difference spectra of oxidized proteins with those of the free amino acids, is that the combined amino acids may react differently towards oxidizing agents in canparison to their free amino acid counterparts. In order to investigate this it was decided to study, in addition to the difference spectra produced by the oxidation of proteins, the difference spectra formed by oxidation of solutions containing cystine, tyrosine and tryptophan together in the same concentrations as their occurrences in the proteins being examined.

'l'he OXidation of Soluble Proteins 'l'he proteins selected for this investigation - casein, insulin and lysozyme - contain approximately the same range of amino acids as found in keratin yet there are significant differences in their amino acid compositions, particularly in the amounts of cystine, tyrosine and tryptophan as shown in Table 4.2. - 109 -

TABLE 4.2 Amino Acid Compositions of Various Proteins132 Amino Acid Content(%) Protein Tyrosine Tryptophan Cystine Insulin 12.6 12.6 Casein 9. 0 2. 5 o. 7 Lysozyme112 3. 7 a. 3 14. 2 Silk fibroin 10. 0 0.4

Prom the contents of cystine, tyrosine and tryptophan in these proteins, it is clear that they provide extremely useful models for investigating the oxidation of these combined amino acids. The protein solutions were prepared to the same epeci~i­ catione as the amino acid solutions - the solvent system employed being a modified Universal buffer comprising of phosphoric, acetic and boric acids yielding a pH of 2. The protein concentrations used were as follows: casein 0.05% insulin 0.1% lysozyme The oxidation of these protein solutions was carried out in the same manner as described earlier for the amino acids. The resulting difference spectra were recorded on the Beckman D.K.2 or Cary 15 double beam spectrophotometers. - 110 -

1. The oxidation of insulin. The difference spectra produced by the oxidation of insulin with potassium permanganate, chlorine, sodium persulphate, peracetic and permonosulphuric acids are shown in Figures 4.33 to 4.37. It is evident from a cursory examination of these spectra, that their structure shows considerable variation and depends on the particular oxidant employed. (1) Oxidation by potassium permanganate The difference spectra formed by the oxidation of insulin with potassium permanganate are shown in Figure 4.33. These spectra are characterized by positive optical densities below 340 mµ. , by a pronounced maxima at 291 mJ.L and a broad minimum between 260 and 280 lDJ.l; in addition there is a slight shoulder at 281. 5 m)l • The overall shape of these difference spectra bears an extremely close resemblance to that formed by the oxidation of tyrosine with the same oxidant (Figure 4.21). The wavelength positions of the insulin difference spectral features do not exactly match those of the difference spectrum of the oxidized free tyrosine, since they occur at slightly longer wavelengths but these variations may be accounted for by the bathochranic shift of the absorption characteristics which is observed when the chranophoric amino acids are incorporated into polypeptide chains. Thus, it may be concluded that the difference spectrum of permanganate FIG. 4. 33 - Ultraviolet difference spectra formed by potassium permanganate oxidation of insulin for 20 minutes at room temperature. 0.5 e

0.4

>- 0.3 ....+> Ul i:: 0.2 G) 'O ...-4 ell 0. 1 ....u +> p.. 0 0 -0.1

-0.2 240 260 280 300 320 340 Wavelength (mj"-), Curves: (a) 25 _,&,1-l.; (b) 50 ),.d.; (c) 75 _µ.l.; (d) 100 _,,ul.; (e) 125 µl.; of 0. lN potassium permanganate.

FIG. 4. 34 - Ultraviolet difference spectra formed by oxidation of insulin chlorine for 30 mins. at room temperature.

0.5 d 0.4 >- ....+> Ul 0.3 i:: G) 'O ...-4 0.2 ell u .... 0. 1 +>p.. C 0 0

-0. 1

240 260 280 300 320 340 Wavelength (mfl, ).

Curves: (a) 10),Ll.; (b) 20,..Ml.; (c) 30;d,; (d) 40_µ.l.; (e) 50.,,u.l.; of 0. lN chlorine.

.-4 .-4

"C "C

0 0

......

......

+,> +,>

_e-

nl nl

p.. p..

CD CD

i::: i:::

Cl) Cl)

u u

-0. -0.

0.2 0.2

0. 0.

1 1

0 0

1 1

FIG. FIG.

240 240

Curves: Curves:

4. 4.

35 35

-

(a) (a)

Ultraviolet Ultraviolet

40µ.l.; 40µ.l.;

260 260

persulphate persulphate

(b) (b)

difference difference

60)A,l.; 60)A,l.;

for for

280 280

10 10

Wavelength Wavelength

spectra spectra

(c) (c)

minutes minutes

80.141.; 80.141.;

formed formed

at at

(m_,,u.). (m_,,u.).

300 300

70°c. 70°c.

(d) (d)

by by

100141., 100141.,

oxidation oxidation

of0.lNsodiumpersulphate. of0.lNsodiumpersulphate.

320 320

of of

insulin insulin

with with

sodium sodium 340 340 8

......

:a 'O

+>

....

'O

.....

....

«I 0 +>

co

Q)

>,. u i::::

+>

>-0. co i:::: «I Q)

p..

u

0.3

0.2

O.

1

0

1

0

a

b

Curves:

260

260

(a)

0.

FIG.

FIG.

e

1 N

40,)4-1.;

permonosulphuric

4.

4.

36

37

-

(b)

-

280

Ultraviolet

280

with

Ultraviolet

Wavelength

80.,.u.1.;

oxidized

Wavelength

,eeracetic

acid.

(c)

difference

insulin

difference

(InJL

120~1.;

acid

300

(m

) •

(10

for

spectra

).

mi!].~S·

spectra

(d)

10

Curves:

minutes

160

7Q

formed

320

320

,,ul.

0

for

c).

O.

(a) (c)

;

at

permonosulphuric

lN

(e)

80

40

by

70°c.

peracetic

JA-1.

J-A--1.

oxidation

200_,,,...... 1.,

; ;

340

(b) (d)

340

acid.

60

100

of

of

.,,u--1.

insulin

,,v-

acid

1.,

;

of - 111 - oxidized insulin arises almost exclusively from the oxidation of the tyrosyl residues of this protein. This view was con:firmed by examining the difference spectra (Pigure 4.38) arising from the permanganate oxidation of a solution of a mixture of amino acids, the concentrations of which correspond to their composition of 0.1% insulin. It is clear that these difference spectra resemble closely that of oxidized insulin and are identical to those produced by the oxidation of free tyrosine alone by potassium per- manganate. It may be concluded from these observations that the oxidations of free and combined tyrosine by potassium permanganate are very similar. The oxidation of insulin, in the presence of high concentrations of sodium chloride, by potassium permanganate could not be investigated by this technique since the sodium chloride caused the precipitation of the protein. (11) Oxidation by chlorine. The difference spectra produced by the oxidation of insulin with chlorine are presented in Figure 4.34. The spectra show a gradual increase in optical densities below 340 mJ.l. but are devoid of any characteristic features. It can be seen that these spectra show no similarity to those produced by the oxidation of free tyrosine with chlorine (Pigure 4.23) and therefore, it appears that the tyrosyl residues of this protein are not attacked initially by this FIG. 4. 38 - Ultraviolet difference spectra formed by the oxidation of a free amino acid mixture corresponding to insulin with potassium permanganate for 30 minutes at room temP_erature. 0.3

>- ...... al i:: a, iO.lr-:___ b a ~ e 0

0

-0. 1 260 280 300 320 340 Wavelength (m_f- ).

Curves: (a) 10_µ.l.; (b) 20JA-l.; (c) 30p.l.; (d) 40_µ.l.; (e) 50_µ.l., of0.lN potas siurn permanganate. - 112 - oxidant. The difference spectra produced by the action of chlorine on a solution of the free amino acids corresponding to 0.1% insulin are shown in Figure 4.39 and also indicate that no attack on the tyrosine occurs. The fact that the absorp­ tivities of the difference spectra of the oxidized protein are much greater than those of a mixture of the free amino acids or cystine alone, suggests that either the absorptivity of the protein molecule has been altered due to aggregation during oxidation or that other groups are also involved in the oxidation. (111) Oxidation by sodium persulphate. The difference spectra formed by the oxidation of insulin by sodium persulphate after ten minutes at 70°0 are shown in Figure 4.35. These difference spectra are characterized by positive optical densities below 340 mJ.L, by maxima at 282 and 289 mp. and by minima at 278 and 284 mJJ.. The close similarity in the difference spectra of persulphate oxidized insulin and tyrosine indicates that the main site of the oxidation of this protein is at the tyrosyl residues. An examination of the difference spectra, shown in Figure 4.40, produced from the persulphate oxidation of a solution of cystine and tyrosine (in the same concentrations as their occurrence in 0.1% insulin) indicates that the spectra produced are almost identical to those from tyrosine only. FIG. 4. 39 - Ultraviolet difference spectra formed by oxidation of a free amino acid mixture corresponding to insulin with chlorine for 30 minutes at room tem£erature. 0.2

~ o. 1 .... a,

=G) "tl ~ a b ...."'u .... I a 8 0

-0.1

260 280 300 320 340 Wavelength (m)-4-).

Curves: (a) S0JA,l.; (b) 40,µ.l., of0.lNchlorine.

a' a'

-~ -~ ......

-a -a

-~ -~

......

......

Ill Ill

u u

Q> Q>

i:: i::

co co

>, >,

0. 0.

0.2 0.2

FIG. FIG.

0 0

1 1

Curves: Curves:

4. 4.

40 40

-

(a) (a)

for for

Ultraviolet Ultraviolet

cystine cystine

260 260

80_µ..l.; 80_µ..l.;

10 10

minutes minutes

and and

. .

(b) (b)

difference difference

tyrosine tyrosine

160JL1.; 160JL1.;

at at

280 280

Wavelength Wavelength

70 70

0 0

corresponding corresponding

c. c.

spectra spectra

(c) (c)

200_.f-ll., 200_.f-ll.,

(~ (~

formed formed

300 300

). ).

to to

of0.lNsodiumpersulphate. of0.lNsodiumpersulphate.

by by

insulin insulin

oxidation oxidation

with with

320 320

sodium sodium

of of

a a

mixture mixture

per per

sulphate sulphate

340 340 of of - 113 -

The evidence suggests, therefore, that the oxidation of free and combined tyrosine by sodium persulphate occurs in a similar manner, in each case modification of the tyrosyl residue occurs in preference to the cystyl residue. (iv) Oxid~tion by peracetic acid. The difference spectra produced by the oxidation of insulin with peracetic acid for ten minutes at 70°0 are presented in Figure 4.36. These spectra possess positive optical densities below 340 lllJ.L and are characterized by a broad maximum at 290 mµ.., a minimwn at 286 m_µ and by a shoulder at 278 m.JJ.. The shape of these difference spectra are unlike those produced by the peracetic acid oxidation of cystine (Figure 4.30) and furthermore, they do not resemble the difference spectra formed by the peracid oxidation of a mixture of cystine and tyrosine, in the same proportions as their occurrences in 0.1% insulin, as shown in Figure 4.41. The difference spectra of peracetic acid oxidized insulin cannot be accurately compared to those produced by treating tyrosine with the same oxidant, since the difference spectra of tyrosine in this case possess insignificant absorption, although there is some indication of fine structure between 270 and 290 m.J.l.. There is a possibility, therefore, that part of the difference spectra generated by the peracid oxidation of insulin is formed by the oxidation of tyroeyl residues indicating that the bound tyrosine is more susceptible

0 0

•.-4 •.-4 "C "C

~ ~

•.-4 •.-4

......

......

~ ~

ll. ll.

(J (J

i::: i:::

Cl) Cl)

CD CD

>, >,

0. 0.

0.2 0.2

0 0

1 1

and and

FIG. FIG.

Curves: Curves:

tyrosine tyrosine

4. 4.

41 41

(a) (a)

-

260 260

80)-ll.; 80)-ll.;

Ultraviolet Ultraviolet

corresponding corresponding

(b) (b)

Wavelength Wavelength

difference difference

100),d.; 100),d.;

280 280

to to

insulin insulin

of of

spectra spectra

(m_µ. (m_µ.

0. 0.

with with

IN IN

). ).

peracetic peracetic

300 300

per per

formed formed

acetic acetic

by by

acid. acid.

acid acid

oxidation oxidation

for for

320 320

10 10

of of

minutes minutes

a a

mixture mixture

at at

340 340

70° 70°

of of

c. c. cystine cystine - 114 -

to oxidation under these conditions than the free amino acid. (v) Oxidation by permonosulphuric acid. The difference spectra produced by treating insulin with permonoeulphuric acid for ten minutes at 70°0 are shown in Figure 4.37•. Clearly these spectra possess low positive absorptivities below 340 mJ.L with a suggestion of fine structure between 270 and 290 m)J.. However, the oxidation of a solution of the free amino acids corresponding to insulin did not yield significant difference spectra and therefore, it was concluded that permonosulphuric acid has very little effect on the tyrosyl residues of insulin under the experi­ mental conditions employed. 2. The oxidation of casein. The ultraviolet difference spectra produced by the oxidation of the protein casein with potassiwn permanganate, chlorine, sodium persulphate, peracetic acid and permono­ sulphuric acid are shown in Figures 4.42 to 4.46. It is evident from a superficial inspection of these spectra, that they are not all alike and therefore, a detailed description of these spectra and the possible factors responsible for them will now be presented. (1) Oxidation by potassiwn permanganate. The difference spectra produced by the oxidation of casein with potassium permanganate are shown in Figure 4.42. The difference spectra are characterized by positive optical FIG. 4. 42 - Ultraviolet difference spectra formed by oxidation of casein with potassium permanganate for 20 mins. at room temperature. 0.6

0.5 ....~ 0.4 ID i:= ~ o. 3 .-4 ·I'd 0.2 ....u O~ 0.1 ~ 6 0 a

-0.1

240 260 280 300 320 340 Wavelength (m.,.u. ). Curves: (a) 10 J-1-1.; (b) 20 .,M-1.; (c) 30 )'4-1.; (d) 40 fA-1.; (e) 50)-41., of 0. lN potassium permanganate. FIG. 4. 43 - Ultraviolet difference spectra produced by the action of chlorine on casein for 20 minutes at room temperature. 0.6

0.5

...... >- 0.4 ID ~ 0.3 'tl ';l o. 2 ...... u o' o. 1 oL----~~2./.~~~~

-0.1

240 260 280 300 320 340 Wavelength (m)J-, ). Curves: (a) 20 j>-1.; (b) 40 .,M-1.; (c) 60 fA-l.; (d) 80 f-'-1.; (e) 100.fA- l., of 0. IN chlorine. FIG. 4. 44 - Ultraviolet difference spectra produced by the oxidation of casein with sodium per sulphate for 10 minutes at 70° c.

0.3

e

0.2 ~ >- +> •.-4 m J:: Q) "d .-4 ~ 0. 1 •.-4 a +> e s ~ ~~~ a - a

0

-0.1

240 260 280 300 320 340 Wavelength (m}'l-).

Curvee: (a) 20_..u. 1.; (b) 40.,...._l.; (c) 60,,...._l.; (d) 80~1.; (e) JOO,._..!.• of 0. JN •odium J> .. r•ulJ>h•~•-

8' 8'

~ ~

......

...... 'O 'O

+> +> ......

Ul Ul

C: C:

CJ CJ

QJ QJ

>, >,

-0. -0.

0.2 0.2

0. 0.

1 1

1 1

01 01

FIG. FIG.

Curves: Curves:

4. 4.

45 45

(a) (a)

::::--,,.,_ ::::--,,.,_

260 260

-

60µ1.; 60µ1.;

Ultraviolet Ultraviolet

E,eracetic E,eracetic

-<-: -<-:

(b) (b)

acid acid

100_,.ul., 100_,.ul.,

difference difference

280 280

~ ~

Wavelength Wavelength

for for

L L

10 10

a a

of of

;;,>-

minutes minutes

spectra spectra

0. 0.

_,.. _,..

(m..,u.). (m..,u.).

lN lN

300 300

peracetic peracetic

produced produced

at_]0~ at_]0~

0 0

b b

acid. acid.

by by

the the

320 320

oxidation oxidation

of of

casein casein

340 340 with with

......

......

0 0

"d "d

-~ -~

......

nl nl

P. P.

i:: i::

Ul Ul

u u

Q) Q)

>-

-0.1 -0.1

O. O.

0.2 0.2

1 1

0 0

FIG. FIG.

a a

--

4. 4.

Curves: Curves:

46 46

....___ ....___

260 260

b b

-

permonosulphuric permonosulphuric

Ultraviolet Ultraviolet

(a) (a)

100_µ.l.; 100_µ.l.;

Wavelength Wavelength

280 280

difference difference

(b) (b)

acid acid

150)-ll., 150)-ll.,

spectra spectra

(m_µ.). (m_µ.).

for for

300 300

10 10

of of

minutes minutes

produced produced

0. 0.

lN lN

permonosulphuric permonosulphuric

at at

by by

70° 70°

320 320

the the

c. c.

oxidation oxidation

acid. acid.

of of

340 340

casein casein with with - 116 - densities, commencing between 287 and 293 D1JJ. and extending beyond 340 mJL as well as below 271 mJL, and by negative optical densities between 271 and 290 mJJ. approximately. With small amounts of added oxidant (10 to 20)-ll) the difference spectra resemble those formed by the permanganate oxidation of tryptophan, except that the difference minimum at 288 mp.. for oxidized tryptophan appears more as a shoulder at 290 mJJ. for the difference spectra of the protein. As more oxidant is employed, the shouldirat 290 mJJ- becomes less distinctive and the positive spectral optical densities cormnence to extend below 293 m;.t. • These results appear to indicate that the tryptophyl residue is the main site of attack by potassium permanganate on casein, although there is some evidence suggesting that the tyrosyl residues are also attacked. This view is supported by the fact that the difference spectra of perman­ ganate oxidized tyrosine have a maximum of positive optical density at 289 ~ and therefore, as the amount of tyrosyl residues attacked increases, the shoulder at 290 m)l. would be expected to decrease and this is what is actually observed in Figure 4.42. This behaviour is exhibited in more detail in the difference spectra generated by the permanganate oxidation of a solution containing cystine, tyrosine and tryptophan in the same concentrations as their occurrence in 0.06~ casein (Pigure 4. 47 ). FIG. 4. 47 - Ultraviolet difference spectra formed by oxidation of a mixture of tyrosine, tryptophan and cystine, corresponding to casein, with potassium perman­ ganate for 20 minutes at room temperature.

0.4

0.3 ...... >, a Ul ~ 0.2 fi Q) a "O ...... 0. 1 nl ....u ....p. 0 0 -0. 1

-0.2

240 260 280 300 320 340 Wavelength (m_µ.).

Curves: (a) 10µ1.; (b) 20.)Ll.; (c) 30_µ.1.; (d) 40_µ 1.; (e) 50.,;vl,1., of 0. IN potassium permanganate. - 116 -

These results, therefore, indicate that the main residue of casein affected by the oxidation with potassium permanganate is tryptophyl, although some oxidation of tyrosyl residues also occurs. (11) Oxidation by chlorine. The difference spectra formed by the oxidation of casein with chlorine are shown in Figure 4.43. Clearly, with low amounts of added oxidant (20).l.l) the difference spectra of chlorinated casein closely resemble that of chlorinated tryptophan (Figure 4.4) as evidenced by its possession of positive optical densities above and below 296 and 266 mJJ. respectively and of negative optical densities between these two wavelengths, characterized by a minimum at 280 mJ1. and a shoulder at 288 mp.. It is, therefore, reasonable to conclude that this difference spectral feature of oxidized casein arises chiefly from the oxidation of its tryptophyl residues. As the amount of oxidant is increased, the differ- ence spectra of casein show a decreasing absorptivity in the negative absorption region until at 80JA.l of added oxidant, the difference spectra are positive at all wavelengths between 340 and 240 MJ.l, whereupon a new spectral feature in the form of a maximum now appears at 288 MJ.L•

A study of the difference spectra produced by the chlorination of tryptophan alone, at concentrations correspond­ ing to its occurrence in 0.05~ casein, has shown that this - 117 - type of behaviour can also occur as evidenced by the spectra in Figure 4.48. The difference spectra illustrated indicate that tryptophan is oxidized by chlorine through a number of stages leading ultimately to a product which yields a differ­ ence spectrum resembling that of chlorinated tyrosine, except that the spectral fine structure occurs at much higher wave­ lengths. The ultraviolet difference spectra of chlorinated casein, therefore, could be attributed entirely to the oxidation of the tryptophyl residue except for the occurrence with high amounts of added oxidant of the distinct maximum at 288 m_µ , since this feature is not evident in the difference spectra of chlorinated tryptophan. The fact that this feature corresponds closely to the difference maximum at 286.5 mJl. for chlorinated tyrosine suggests that this feature of the difference spectra of oxidized casein results from a subsequent attack on the tyrosyl residues after most of the tryptophyl residues have undergone oxidation. The slight variations in wavelength positions of the maxima for free and combined tyrosine could arise from the bathochromic shift associated with the incorporation of the free amino acid into a poly­ peptide chain. The difference spectra produced by the action of chlorine on a solution containing cystine, tyrosine and tryptophan in the same proportions as their occurrences in 0.05% casein are 'O .-4 ......

0

~

>-

Q) ~

nl

u

-0.1

0.2

0.1

0

I L

,,.,,

Curves:

240

FIG.

e d

f,h

(a) (g)

-

4.

8g.,u_ 5_,.ul.;

48

1.;

-

260

Ultraviolet

(b)

corresponding

(h)

10µ.1.;

100_µ1.,

Wavelength

difference

(c)

of

280

to

0.

15,µ1.;

its

lN

(mJ,A-

concentration

chlorine.

spectra

(d)

~

).

20_µ.1.;

formed

300

§

f

e

in

(e)

by

0.

05%

oxidation

40_µ.1.;

casein,

320

(f)

01

with

tryptopnan,

60_µ.1.;

chlorine.

340 - 118 - shown in Figure 4.49. Clearly, these spectra exhibit the same type of behaviour as discussed above except here the spectral features are more clearly resolved. At high additions of chlorine (>40).ll) these spectra exhibit a maximum at 285.5 m)l, corresponding very closely to the maximum at 286.5 mJL for chlorinated tyrosine and consequently, it is thought that this feature in the spectra of oxidized casein and of a mixture of cystine, tyrosine and tryptophan corresponding to casein, arises from the oxidation of the tyrosine entity, subsequent to an initial preferential attack on the tryptophan and cystine components. (iii) Oxidation by sodium persulphate. The difference spectra generated by the oxidation of casein with sodium persulphate for ten minutes at 70°c are shown in Figure 4.44. Clearly, the shape of these difference spectra is almost identical to those formed by the oxidation of tryptophan by sodium persulphate (Figure 4.7). For compari­ son purposes the difference spectra, produced by the action of sodium persulphate on a solution containing cystine, tyrosine and tryptophan in the same concentrations as their occurrences in 0.06% casein, are shown in Figure 4.50. It is clear that the structure of these difference spectra resemblesvery closely those formed by the action of persulphate on tryptophan alone and on the protein casein. These results, therefore, suggest that the main site of FIG. 4. 49 - Ultraviolet difference spectra formed by oxidation of a mixture of tyrosine, tryptophan and cystine, corresponding to casein, with chlorine for 20 minutes at room temperature.

0.5

0.4 >, +J •.-4 CD 0.3 i::: Cl) 'C 0.2 RI -u •.-4 o. 1 +J ll. 0 0

-0.

240 260 280 300 320 340 Wavelength (mj.4-).

Curves: (a) 20µ1.; (b) 40µ1.; (c) 60pl.; (d) 80_,,u.1.; (e) 100_,M-1., of 0. lN chlorine.

.-4 .-4 t

"C "C 0 0

......

......

P.. P..

~ ~

u u

-0. -0.

0.1 0.1

0.2 0.2

0.3 0.3

0 0

1 1

FIG. FIG.

i i

~ ~

240 240

Curves: Curves:

___:\\ ___:\\

4. 4.

50 50

-

'\\ '\\

0. 0.

(a) (a)

Ultraviolet Ultraviolet

tyrosine, tyrosine,

sodium sodium

lN lN

40.JLl.; 40.JLl.;

260 260

sodium sodium

persulphate persulphate

tryptophan tryptophan

difference difference

(b) (b)

persulphate. persulphate.

Wavelength Wavelength

S0JA-1.; S0JA-1.;

280 280

for for

and and

spectra spectra

10 10

(c) (c)

cystine, cystine,

(m,,u.). (m,,u.).

minutes minutes

120 120

produced produced

;,c-1.; ;,c-1.;

corresponding corresponding

300 300

at at

70°c. 70°c.

(d) (d)

by by

160/A-l.; 160/A-l.;

oxidation oxidation

b b

d d

e e

a a

C C

to to

320 320

casein, casein,

(e) (e)

of of

a a

200_,.ul., 200_,.ul.,

mixture mixture

with with

of of

of of 340 340 - 119 - attack by sodium persulphate is the tryptophyl residue since there is no spectral evidence which could be attributed to oxidative modification of the tyrosyl or cystyl residues. (iv) Oxidation by peracetic acid. The difference spectra produced after treating casein with peracetic acid for ten minutes at 70°0 are shown in Figure 4.45. These difference spectra possess very low positive optical densities above and below 296 and 264 lllJ.l respectively and negative optical densities between these two wavelengths. In this respect these difference spectra resemble those produced by the oxidation of tryptophan by peracetic acid (Figure 4.12) but because of the extremely low absorptivities, both positive and negative, very little fine spectral structure can be detected. It seems likely, therefore that the difference spectra formed may in part arise from the oxidation of the tryptophyl residues of caaein but this does not undergo modification to the same extent as that produced by the other oxidants already examined. From the lack of reactivity between tyrosine and peracetic acid and from the known affinity of this oxidant for cystine44, it is thought that the oxidation occurs primarily with the cystyl residues of casein. (v) Oxidation by permonosulphuric acid. The difference spectra generated by treating casein with permonosulphuric acid for ten minutes at 70°0 are shown in - 120 -

Figure 4.46. These spectra show a gradual increase in optical density below 340 m_µ but their low absorptivity and lack of structure indicate that there is little modifica­ tion of the tryptophyl or tyrosyl residues of this protein by this peracid. 3. The oxidation of lysozyme. The ultraviolet difference spectra produced by the oxidation of lysozyme with potassium. permanganate, chlorine, sodium persulphate, peracetic and permonosulphuric acids, are shown in Figures 4.51 to 4.55. It is clear that these difference spectra exhibit some variation in their absorption characteristics depending on the oxidant employed and therefore, the spectra generated by each oxidant will be described in more detail. (1) Oxidation by potassium. permanganate. In Figure 4.51 are presented the difference spectra formed by the oxidation of lysozyme with potassium. permanganate. Clearly, the shape of this series of difference spectra bears a striking resemblance to those obtained by the oxidation of tryptophan alone with potassium permanganate (Figure 4.2). With increasing amounts of a~ded oxidant, it can be noted that both the positive and negative components of the difference spectra increase without sigl!ificant alteration in the overall structure of these spectra. This behaviour implies that the oxidation -

-~ 0 ~

-~

.....

P.

ro

i:::

U)

>­

-0. -0. -0.3 -0.4

0.4

0.3

0.2 o.

Curves:

1

0

2

1

~------U,.+---il----

FIG.

C

(a)2.opl.

4.51

of

0.

260

lN

-

·;

Ultraviolet

assium

at

potassium

Wavelength

by

(b)

room

oxidation

40_µ..

280

permanganate

tem_perature.

l.;

permanganate.

300

difference

(m_µ..).

of

(c)

lysozyme

60.fA-

2 340 320

l.;

for

spectra

with

20

mins.

pot-

formed

-

"Cl

0 ....

....

.....

.....

ro

U)

P.

CU i:::

u

>-

-0.7

-0.5

-0.6

-0.4

-0.2

-0.3

-0.

0,2~~

O.

0.3

0. 0

• 5

0

4 1

1

Curves

:(a)

of

260

FIG.

ion

spectra

20

20)'-'-

0.

lN

mins.

of

Wavelength

l.

4.

chlorine.

lysozyme

280

;

52

resulting

(b)

at

-

room

40_,u.

300

C

Ultraviolet

by

(~).

1.

from

b

temE_erature.

320

chlorine

;

(c)

the

340

difference

60.}A-

oxidat­

for

1.

, FIG. 4. 53 - Ultraviolet difference spectra. Persulphate oxidation of lysozyme for 10 minutes at 70°c.

0.5

0.4

0.3

0.2 0. 1 ...... >, Ill 0 i::: a, 'tJ -0. 1 .-4 RI ...... (J -0.2 P. 0 -0.3

-0.4

-0.5

240 260 280 300 320 340 Wavelength (m)A- ).

Curves: (a) 5.,u.l.; (b) 10 J-Ll.; (c) 15 p.l.; (d) 20,,u.l., of 0. IN sodium persulphate. t

..... 'tl

-~ ....

....

..... 'tl

t

....

...

...

...

nl

u

rn >-

Cl,)

~

nl 11) >-

Cl,)

~

u

-0.

0.2

0.

0.

1

1

0

0

1

FIG

240

240

FIG.

200

4.

54

_µ.

4.

l

-

55

oxidant

Ultraviolet

at

-

260

260

Ultraviolet

70°c.

lysozyme

difference

difference

for

Wavelength

280

280

10

spectra.

minutes

spectrum.

(m_fA-

Per

at

70°c.

300

300

acetic

).

Permonosulphuric

acid

fj

d

e

a

oxidation

320

320

Curves

acid.

(c) (e)

200_,,u.

120.,u.l.;

of

acid

lysozyme

:

(a)

1.

oxidation

,

of 40

(d)

340

340

_µ.

0.

for

1.

160_µ1.;

IN

;

of

10

per

(b)

mins.

acetic

8 0

_,,....

1.

; - 121 - of this protein is proceeding in the same manner throughout. Prom this and from the fact that there is no feature in these difference spectra that can be attributed to the oxidative modification of the tyroayl residues, it appears that the initial attack of permanganate on lysozyme occurs preferentially at the tryptophyl residues. (11) Oxidation by chlorine. The difference spectra produced by the oxidation of lysozyme with chlorine are shown in Pigure 4.52. Once more, it is evident that the structure of these difference spectra is very similar to that of the difference spectra formed by the action of chlorine on tryptophan alone (Figure 4.4), as evidenced by the fact that they both possess the same spectral features, such as positive optical densities above and below 298.5 and 266 m.J.l respectively and minima at 282.5 and 290.5 mJJ., both of negative optical density. Since the difference spectra do not undergo significant alteration in structure with increasing amounts of added oxidant, it appears that the attack of chlorine on lysozyme is mainly on the tryptophyl residues. However, in view of the fact that polarographic studies (Chapter III) showed that chlorine attacks cystine and tryptophan simultaneously, the possibility of attack on the cystyl residues cannot be overlooked. (111) Oxidation by sodium persulphate. The difference spectra generated by treating lysozyme - 122 - with sodium persulphate at 70°C for ten minutes are shown in Figure 4.53. Clearly, this aeries of difference spectra bears a very strong resemblance to those generated by the action of sodium persulphate on tryptophan alone (Figure 4.7). The fact that t~e shape of these difference spectra remains virtually unaltered with increasing amounts of added oxidant, suggests that the reaction is proceeding in the same manner throughout. It may be concluded, therefore, that the main site of lysozyme which 1s attacked by sodium persulphate is the tryptophyl residue, for there is no spectral evidence for the oxidative modification of the tyrosyl or cystyl residues. (iv) Oxidation by peracetic acid. In Figure 4.54 are presented the difference spectra produced by the oxidation of lysozyme with peracetic acid for ten minutes at 70°c. A feature of this series of difference spectra is the low positive and negative absorptivities. The structure of this series of difference spectra remains unaltered with increasing amounts of added oxidant and it is characterized by a broad maximum, of positive optical density, at 299 MJL and by the minima at 284 and 292.6 mp. of negative optical density. These spectral features are somewhat similar to those produced by the oxidation of tryptophan only by this oxidant (Figures 4.10 to 4.12). The fact, however, that the shapes of the difference spectra of the oxidized lysozyme and of oxidized tryptophan are not identical indicates - 123 -

that some other residues, apart from tryptophyl, are being modified, probably the cystyl. (v) Oxidation by permonosulphuric acid. The difference spectrum produced by the oxidation of lysozyme with p~rmonosulphuric acid for ten minutes at 70°0 is shown in Figure 4.56. It is clear fran the low absorptivi­ ties of this difference spectrum that there is no significant modification of the aromatic amino acid residues of this protein. However, there is some fine spectral features apparent, such as the positive optical densities above and below 294 and 273 m respectively, and minima at 283 and 292.6 m of negative optical density. The overall shape of this difference spectrum and the wavelength positions of the fine spectral structure resemble that formed by the oxidation of tryptophan. It appears, therefore, that under the experi­ mental conditions employed the tryptophyl residues of lysozyme are slowly oxidized by permonosulphuric acid. (vi) General conclusions. The preceding investigation has yielded usef'ul data concerning mechanisms of oxidative modification of certain soluble proteins and the results may be SUJID'llarized briefly as follows: (a) The oxidation of proteins containing cystine and tyrosine but no tryptophan, such as insulin, may proceed either

by preferential oxidation of the tyrosyl residues or of the - 124 - cystyl residues depending on the oxidant employed. Thus the former behaviour appears to occur with potassium permanganate and sodium persu.lphate whilst the latter is thought to occur with chlorine, peracetic and permonosulphuric acids. (b) For p:r-oteins containing tyrosine and tryptophan and only minute quantities of cystine, such as casein, there appears to be preferential oxidation of the tryptophyl residues with potassium permanganate, sodium persulphate and chlorine. Prom the known reactivity of the peracids with cystine and from the lack of spectral evidence indicating significant modification of the aromatic amino acid residues, it is likely that the oxidation of casein by these oxidants is confined chiefly to the cystyl residues. (c) It is found that, for proteins possesel.ng tyrosine, tryptophan and cystine such as lysosyrne, potassium permanganate, sodium persulphate and chlorine readily attack the tryptophyl residues, although in the latter case there is polarographic evidence suggesting that the cystyl residues are oxidized concurrently. The oxidation of lysozyme by the peracids is thought to be chiefly confined to the cystyl residues although there are indications that tryptophyl residues are affected to a small degree. The Oxidation of Fibrous Proteins The main problem associated with the investigation of fibrous proteins is the relative insolubility of these canpounds - 126 -

in simple solvents. Silk fibroin, for example, can be dissolved in concentrated solutions of inorganic salts, notably the thiocyanates of lithium, sodium and calcium133, but these solutions usually preclude the application of spectroscopic techniques (or other physico-chemical techniques) since the high concentrations of these salts affect the analyses. In the case of wool, dissolution is not possible without extensive degradation of the keratin. It was thought, however, that the technique of ultraviolet difference spectrscopy could be applied by first oxidizing the fibre and then hydrolyzing it in solutions which cause the least amount of amino acid degradation and finally canparing this with a standard solution prepared by hydrolyzing an equal weight of unmodified fibre by the same procedure. 1. The oxidation of silk fibroin. The oxidation of silk fibroin by potassium permanganate and chlorine was investigated by employing the following procedure: 25 mg. samples of silk were accurately weighed and placed in 25 ml. volumetric flasks and to.these were added 1 ml. of a solution of Ultravon J.U., a non-ionic wetting agent and 10 ml. of 30% sulphuric acid. The required amount of decinormal oxidant was then introduced and the reaction was allowed to proceed until the permanganate was decolourized or no odour of chlorine could be detected, after which the silk was - 126 - hydrolyzed for 12 hours at 105°c. The volumetric flasks were then allowed to cool and made up to volume with distilled water. The difference spectra of these samples were then obtained by comparing them to a hydrolysate of unmodified silk prepared ~Y the same procedure described above. (1) Oxidation by potassium permanganate. The ultraviolet difference spectra of silk oxidized by potassium permanganate are shown in Figure 4.55. These difference spectra possess positive optical densities below 340 mJJ. and they are characterized by a broad maximum at 292. 5 mµ. and by a minimum at 274 m.JJ. in addition to a shoulder at 280 ~- The shape of the minimum feature together with the accompanying shoulder is almost identical to the mirror image of the absorption spectrum of tyrosine and furthermore, the difference minimum and accompanying shoulder occur at precisely the same wavelengths as the absorption maximum and shoulder of normal tyrosine. It is reasonable to conclude, therefore, that this feature of the difference spectra is an "abstraction" spectrum arising from the modification of part of the original tyrosine in the oxidized sample. The difference spectral component of positive optical densities above 290 mJJ. probably arises from the products of the oxidation of the tyrosyl residues. (11) Oxidation by chlorine. The difference spectra produced by the action of chlorine FIG. 4. 56 - Ultraviolet difference spectra of silk oxidized by potassium permanganate.

0.3

0.2

>- ....+,I m s:: ~ 'O .-4 0.2

...."'u d +,I ~ C p,. e 0 -~~ b a I '- ~ 0

-0. 1 240 260 280 300 320 340 Wavelength (m.,u. ).

Curves: (a) 0. 25 ml.; (b) 0. 50 ml.; (c) 0. 75 ml.; (d) 1. 00 ml.; (e) 1. 25 ml., of 0. lN potassium permanganate. - 127 - on silk are shown in Figure 4.57. Once again these spectra are characterized by a minimum feature with an accompanying shoulder at 274.5 and 280 m_µ. respectively indicating a loss of tyrosine while the positive optical densities, above 290 mµ, of the difference spectra may be attributed to the products arising from the oxidation of tyrosine. 2. The oxidation of wool keratin. Attempts were made to examine the effects of oxidation of wool by potassium permanganate and by chlorine by employing the technique of ultraviolet difference spectroscopy but these were unsuccessful since no satisfactory method of dissolving oxidized wool without extensive chemical modification was found. If is of interest, however, to consider the results of chemical analyses which have been carried out by other workers during studies of the oxidation of wool by these oxidants. In general, these workers are in agreement about the loss of combined cystine during these treatments but their results concerning the loss of tyrosyl or tryptophyl residues are conflicting. McPhee50 has found that chlorination of wool causes a loss in combined cystine but he was unable to detect any change in the tyrosine or tryptophan content. The latter results are not in agreement with those of Alexander and Gough86, and Graham and Statham84 , who have found that chlorine reduces the tyrosine and tryptophan contents of wool FIG. 4. 57 - Ultraviolet difference spectra. Action of chlorine on silk.

0.8

0.7

0.6

~ ....+> 0.5 ID i::: Q) "Cl 0.4

,-4 RI 0.3 ....u +> p,. 0 0.2

0. 1

0

-0. 1

240 260 280 300 320 340 Wavelength (rn_,µ ).

Curves: (a) 1. 00 ml.; (b) 1. 50 ml., of 0. lN chlorine. - 128 - respectively. In the case of treatments of wool with neutral permanganate in solutions of sodium chloride, KcPhee100 was unable to detect losses in tyrosine or tryptophan, although the cystine content was lowered slightly. These results are fairly consisteµt with those of Andrews, Inglis, Rothery and Williams37 except that these authors did find a slight lowering of the tryptophan content. It is clear, therefore, that the question of preferential attack of the tryptophyl, cystyl or tyrosyl residues of wool cannot be resolved from the results of these workers. On the basis of the preferential attack of oxidising agents on amino acid residues during the oxidation of soluble proteins, and a similar behaviour between insoluble silk and soluble tyrosine, it is tempting to suggest that a similar preferential attack may occur during the treatment of wool with oxidising agents. However, because of the complexity of the morphological structure of wool and its inherent insolubility, the oxidation processes occurring with wool are heterogeneous and, consequently, may be vastly different from those occurring with the soluble proteins. - 129 -

Chapter V: THE FORMATION BY OXIDIZING AGENTS OF FREE RADICALS IN WOOL AND SILK

Introduction

Previous mention was made of the fact that oxidation involves an electron abstraction from chemical species and furthermore, it was shown that the oxidation of organic compounds can occur by three distinct mechanisms: Homolysis. In this case the electron abstraction occurs together with the withdrawal of a proton or another radical,

R - CH3 --~ R - CH2 • + H. Heterolysis. Here the electron-pair constituting the chemical bond is eliminated with one of the bonding atoms or groups and this usually results in the formation of a carbonium ion,

R-CH2 :X --> R-CH2+ + 3. Ionization. This involves electron abstraction from an organic compound and usually necessitates the use of high­ energy irradiation,

+ E. The homolysis and ionization electron-abstraction processes usually produce free radicals, which can be defined as species modified so that they contain an unpaired electron in their electronic structures. Thus, since it is possible to generate free radicals by oxidation, a study of their - 130 - formation in oxidized wool should provide useful data concern­ ing mechanisms of protein oxidation, in particular the sites of initial attack.

Detection of free radicals. Either chemical or physical methods can be used for the detection of free radicals. 1. Chemical methods. The unpaired electron endows the free radical with an abnormally high chemical reactivity which can be used to demonstrate the presence of these species in a system, for example, they are capable of initiating addition polymerization of a vinyl monomer under conditions where polymerization does not ordinarily occur. The chemical methods of detection, however, are not applicable in every case, for example, they are unable to detect the presence of radicals stabilized by steric hindrance. 2. Physical methods. The physical methods do not suffer from any of the disadvantages of the chemical methods, for they depend only on the magnetic moment arising from the uncompensated spin of the free electron. A number of physical methods for radical detection exist such as magneticeuaceptibility, absorption spectroscopy, mass spectroscopy and electron spin resonance spectroscopy and these methods have been reviewed recently by - 131 -

Ingraml34. The method found most suitable for this investi- gation was electron spin resonance (ESR) spectroscopy.

Electron spin resonance spectroscomr Since an electron has an intrinsic spin, it will also possess an angular momentum I~, where I is the moment of inertia of the electron and wits angular velocity. On the basis of quantum mechanical theory, the angular momentum of an atomic particle can assume only integral or half-integral values of h/211' so that:

= nh or ½!!a 2'n' 21T where, h = Planck's constant n = an integer, for an electron this angular momentum becomes½ (h/2rr), so that the spin is equal to i in units of h/211"'• Since a spinning electron can be compared with a current passing through a circular coil, there will be a magnetic field associated with it having the properties of a small bar magnet and this is called the magnetic dipole of the electron. The magnetic dipole moment is a vector quantity, orientated parallel to the spin-axis of the electron, with a magnitude defined as a Bohr magneton. According to quantum mechanics and magnetic resonance theory, an applied magnetic field can produce only two orientations of the spin-axis of the electron. These are - 132 - parallel and anti-parallel to the magnetic field. The two possible orientations of the magnetic dipole moment of an electron under the influence of an external magnetic field give rise to two energy levels; the energy difference between these levels being given by the following equation,

= P., g H (1) where, g = the spectroscopic splitting constant, which is proportional to the external applied magnetic field. = energies of the two orientations of the electron's magnetic dipole moment, anti-parallel and parallel respectively. /3 = Bohr magneton. H = applied magnetic field. The resonance condition follows with the introduction of the Bohr frequency relationship: E2 - E1 = hv (2) where, h = Planck's constant. v = the frequency of radiation associated with the energy-difference.

By equating equations (1) and (2), the following relationship is obtained:

hl) = g ~ H (3) From this equation it can be seen that the frequency of the - 133 - absorbed radiation is directly proportional to the applied magnetic field, or in other words, the resonance can occur at any frequency of the applied radiation provided the magnetic field is of a suitable magnitude. Thus, in theory, a spectrometer can be constructed with a constant direct-current magnetic field and the frequency of radiation is then varied until absorption takes place. In practice it is difficult to carry this out because of the non-flexibility of high-frequency generators. It has been found more convenient to use a constant frequency source of radiation, a klystron, and to vary the magnetic field to obtain the absorption. The ESR spectrometer employed in this investigation operated at 9,470 Mc/sin the X-band region (8,000 - 12,000 Mc/s) of the microwave spectrum and employed 100 kc/a magnetic field modulation to achieve high sensitivity. This resulted in the recording of the first derivative curve of the ESR spectrum. The normal ESR spectrum obtained for a free unpaired electron is shown in Figure 5.1 (a) and the first derivative spectrum is shown in Figure 5.1 (b). Usually in a free radical, the unpaired electron is not "free" but is either associated with the crystalline field incorpo1·ating the electron, or else with the magnetic field arising from adjacent nuclei having an odd number of protons. These associations cause modification of the ESR spectrum (termed hyperfine splitting) am these structural modifications FIG. 5. 1

(a) Normal ESR absorption spectrum of a free unpaired electron.

(b) 1 st derivative ESR curve of a free unpaired electron. - 134 - can be used to identify the molecular enviror.ament of the unpaired electron present. The nature of this hyperfine splitting can be explained on the basis of quantwn mechanical theories. Since the.hyperfine structure of the ESR spectrwn of a free radical ie related to the environment of the unpaired electron then if only one environment exists for the unpaired electron in a molecule, the hyperfine structure will define the electron's environment precisely. If, however, there are two or more different locations for the unpaired electrons in a molecule and if the numbers of electrons associated with each are of approximately the same magnitude, then the hyper­ fine structure obtained will be a resultant of all the structures contributed by the separate electron locations (since all free electrons absorb at the same frequency of radiation). Despite this problem, the structure of the ESR spectrum may still provide useful data concerning the locations of unpaired electrons in a molecule. Before describing some of the experimental work carried out with wool in this connection it may be useful to discuss briefly some previous work carried out on proteins and related compounds.

The formation of free radicals in proteins Most of the work carried rut on the formation of free - 135 - radicals in proteins has been concerned with those generated by ionizing radiation such as X-ray, 15-ray and ultraviolet radiation. Gordy and his coworkers135, 136 have found that the ESR spectra of X-irradiated proteins yield only a few simple resonance patterns, whereas irradiation of the individual amino acids comprising these proteins give rise to different and, in many cases, complex spectra. In the case of X-irradiated keratins, these workers observed that the spectra closely resembled those of X-irradiated cystine or cysteine (Figures 5. 2 (a) and (b) ) but differed from the spectra obtained from the other amino acid components of keratins. The spectral component which is generally attributed to an unpaired electron associated with a sulphur atom is shown in red in Figure 5.2 (a). This component occurs in the low-frequency range of the spectra of both X or 't5-ray irradiated wool and cystine and is well removed from the frequency at which most "free" unpaired electrons absorb, the so-called g = 2.00 position denoted by the arrows in Figures 5.2 (a) and (b). On the basis of the similarity in the spectra, these workers suggested that the unpaired electrons in X-ray irradiated keratins were associated with the sulphur atoms of combined cystine or cysteine, either as a three electron bond between the two sulphur atoms, R-S.t.S-R FIG. 5. 2 - 1st derivative ESR spectra

H

65G

f

(b) X-irradiated wool keratin. - 136 - or as a single electron on one sulphur atom, R - s• It was suggested that although the location of the original radiation attack on the protein was not specific, a transfer of the damage occurred so that finally its location appeared at some "electron sink", such as the sulphur atoms of combined cystine. Evidence supporting this transfer mechanism in irradiated proteins and polypeptides has been obtained by a number of workers137, 138• In the case of X-irradiated proteins containing little or no cystine, such as silk, cattle hide and fish scale, the ESR spectra were very similar, each having the form of a doublet, which normally signifies an interaction between an unpaired electron and one proton. Gordyl36 ascribed this resonance to an unpaired electron located on an oxygen atom and interacting with the nuclear spin of a proton on an adjacent polypeptide chain. In general, Gordyl36 concluded that X-irradiation of proteins produces two basic types of ESR resonance, either similar to irradiated silk or similar to irradiated cystine, while for some proteins the ESR resonance was a combination of these two types. These basic ESR patterns Gordy denoted "cystine-like" and "silk-like"; the former occurring in irradiated proteins of a high cystine content and the latter in proteins deficient in cystine. Kenny and Nicholls139 have - 137 - fowid that ?5-irradiation of wool produces radicals which give "cystine-like" and "silk-like" ESR resonances. The conclusions reached by Gordy et al, however, concernizig the environment of unpaired electrons in X-ray irradiated ker~tins may be criticised on the basis of their assumption that the spectrum of X-ray irradiated keratin is a simple resonance. For example, it has been found that X-ray irradiated polymethyl methacrylate does not yield a single resonance and in fact, its spectrum has been shown to consist of two contributing structures140• It must be noted in this connection that polymethyl methacrylate is a relatively simple high polymer, since it consists of only one identical repeating molecular species. The problem associated with wool may be appreciated when it is realised that wool consists of not one but eighteen or so, different repeating species occurring in unknown sequence in the polYPeptide chains. Thus, conclusions concerning chemical environment of unpaired electrons in wool based only on ESR spectral evidence are highly suspect. :Dlnlop and Nicholls1411 employing the ESR spectroscopic method, have demonstrated that free radicals are formed by irradiating wool with ultraviolet light but they were only able to identify those free radicals associated with the sulphur atoms of the cystyl residues. These workers, however, did obtain some indication that tyrosine and - 138 - tryptophan could contribute to the other free radical environ­ ments. In an earlier investigation, the author142 demonstrated that free radicals could be formed in wool and silk by oxidation and further, the ESR spectra of wool and silk were found to be very similar to the corresponding '"a-irradiated proteins. On this basis it was suggested that the radical sites were alike, implying that oxidation and irradiation produce similar effects in proteins. However, the free radical environments of the protein have not been elucidated unequivocally and therefore, it was decided to examined the ESR spectra of various chemically pre-modified wools and silk to ascertain what effect the premodification has on the hyperfine structure of the ESR spectra.

Experimental procedure The employment of ESR spectroscopy to detect the formation of free radicals in wool keratin and silk fibroin requires that the sample be relatively dry, for water in its liquid phase has a large non-resonant absorption due to the inter­ action of its large dipole moment with the microwave electric field. Hence aqueous samples usually cause large damping and a considerable reduction in sensitivity if inserted into the cavity of the ESR spectrometer. Thus, the following basic experimental procedure was employed in the proceeding i1ivestigatione: - 139 -

Samples of scoured wool, in the form of yarn were extracted with ether and ethanol in a soxhlet apparatus for four hours each to remove residual grease and oil. These were then thoroughly rinsed in many changes of distilled water, centri­ fuged to remove excess water and conditioned for 48 hours in an atmosphere of 65% R.H. maintained at 70°F. The samples (approximately 25 mg.) were next accurately weighed and treated with solutions of oxidant (0.1%) for a predetermined time using a 50:1 liquor to wool ratio. All these solutions were made O.OlN with respect to sulphuric acid and contained 0.1% Lissapol N (•Iv), a non-ionic wetting agent. After treatment the wool was centrifuged at approximately 5,000 r.p.m. for one minute to remove excess oxidant solution and then packed down to the sealed end of a small bore glass tube (internal diameter 4 mm.). The tube was immediately connected by a two-stage mechanical vacuum pump through a liquid-nitrogen vapour trap and the evacuation started just prior to the tube being immersed for 10 minutes in an oil­ bath maintained at 110°0 ! 1°0. At the conclusion of this treatment the tube was sealed, allowed to cool to room temperature and then placed in the cavity of the ESR spectro­ meter where the first derivative of the absorption spectrum was traced out. Repeating the above procedure, except that the oxidant was absorbed on non-protein substrates e.g. alumina, cellulose, yielded no ESR spectra, clearly indicating - 140 - that the free radicals are produced by attack on the protein and not by breakdown of the oxidant. The masses of wool used to obtain the ESR spectra were in excess of the amount required for absorption in all cases and therefore, .only a small part of the samples in the tube interacted with the klystron radiation. Since the wool was packed to approximately the same density each time the absorption spectra should be independent of the total mass used. In the case of 15 -irradiated wool and silk the following procedure was employed: Fibre samples (approximately 0.3 mg.) were accurately weighed and placed to one end of small bore glass tubes (internal diameter 4 mm.) which, to remove traces of air and moisture, were evacuated (through a liquid-nitrogen vapour trap) for

4 days with a two stage mechanical rotary pump backing an oil-diffusion pump. The tubes were then sealed and irradiated with 'lr-rays from the University of New South Wales' 500 curie Co60 source at an approximate does rate of 2 X 106 roentgens per hour. After exposure to the required dose, the ends of the tubes farthest removed from the sample were flame-annealed to remove any radicals formed in the glass by irradiation and after cooling, the wool was shaken down to these ends. The

ESR spectra of the ~ -ray irradiated wool were obtained by - 141 - placing the tube in the cavity of the ESR spectrometer.

Results Preliminary experiments demonstrated that free radicals were generated, in sufficient concentrations to be detected by ESR spectroscopy, during the oxidation of wool only in the case where sodium persulphate was employed as oxidant. Attempts to form free radicals in wool by employing other oxidants such as potassium permanganate, chlorine, peracetic and permonosulphuric acids were unsuccessful. This, of course, does not rule out the possibility that free radicals are produced during these treatments but, clearly, the con­ centrations of free radicals, if produced, must lie below the limits of detection of the ESR spectrometer. It was decided, therefore, to restrict the subsequent investigation to free radicals generated in the fibrous proteins by sodium persulphate oxidation. Attempts to detect by ESR spectroscopy free radicals formed in uncombined amino acids by persulphate oxidation have not been successful and this probably arises from the fact that the reaction is restricted to the surface of the amorphous solid, thereby leading to insufficient numbers of free radicals to be detected by the ESR spectrometer. Consequently, it is not possible to compare the spectru~ of wool with those of the free amino acids as has been done with the radiation-induced - 142 - free rad1calsl36,139. However, because similarities exist in the spectra of free radicals formed in unmodified wool by either irradiation or by oxidation, it was decided to compare the spectra of free radicals formed in chemically premodified wool by either.method in order to determine whether further similarities occurred and to examine what effect the prior chemical modification of certain groups would have on its ESR spectrum. It was found in most cases that the free radicals produced for a particular premodified wool were similar irrespective of their mode of formation, although the ~-ray induced free radicals, in general, usually yielded more highly resolved spectra. The chemical pretreatments employed to modify the wool have been previously reported143,144• In no case was it possible to detect any free radicals in these chemically modified wools. (a) Modification of the disulphide bond in wool. The disulphide bonds of the cystyl residues of wool were disrupted by the following methods: (1) Peracetic acid oxidation. (11) Reduction followed by reaction with iodoacetic acid. (111) Conversion to the thio-ether cross-links of lanthionine. Peracetic acid oxidation of wool mainly converts the combined cystine to cysteic acid residues as shown by the following

equation: [O] > 2 R-CH z-SO ;!I - 143 -

Some partial oxidation products of the disulphide bond may be produced but under the conditions of the treatment they are present in only minor amounts49• Previous results have indicated that very little modification to other groups in wool occurs wi~h this treatment, although it is known that this oxidant can also oxidize tryptophan and methionine40• The modification causes breakdown of the disulphide linkage and involves oxidation of the sulphur atoms in wool to their highest oxidation state (+6). This means that all the electrons in the valency shell are involved in bonding and are no longer free to participate in subsequent oxidation reactions. This, therefore, eliminates subsequent partici- pation of the sulphur of wool in free radical formation, since this would entail a partial oxidation of a fully oxidized atom and this is impossible. The other two modifications of the disulphide bond are not as specific in their reaction with wool as that. with peracetic acid. In the case of reduction of disulphide bonds followed by blocking with iodoacetic acid, the reduction is specific for the disulphide bond in wool as shown, 200 thioglycollic acid> 2 R-CH2-SH

The blocking of the thiol groups with iodoacetic acid is less specific, si11ce there is a possibility that this reagent can combine with the phenolic hydroxyl group of tyrosine, the - 144 - terminal amino groups and the free amino groups of combined lysine. The conversion of the cystyl disulphide cross-link to the thio-ether linkage of lanthionine in wool by treatment with potassium cyanide is also a doubtful specificity. The potassium cyanide solution will be alkaline by hydrolysis and it is known that the action of alkali on wool is an extremely complex process affecting amino acids other than cystine. The cystyl disulphide bond in wool is converted into compounds represented by the following formulae: (i) Reduced and iodoacetylated wool, R-CH2-S-CH2-COOH (11) Lanthionine residues, R-CH2-S-CH2-R It can be seen from these structures that the disulphide bond character of wool has been removed by both these modifications but the sulphur atoms are still in a low oxidation state (+2) and therefore, are capable of further oxidation. In other words, in these two cases the sulphur in wool, although not of disulphide character, is still capable of taking part in subsequent free radical formation by electron removal processes. The ESR spectra of these modified wools, obtained by the usual methods, are shown in Figures 5.3 to 5.6 together with that of unmodified wool. It can be seen that some similarities between the spectra of the modified wools still exist, irrespective of the method used in the formation of the free radicals. FIG. 5. 3 - 1st derivative ESR spectra

H

65G

(a) 'lS -irradiated wool premodified with peracetic acid.

(b) Per sulphate oxidized wool premodified with peracetic acid. FIG. 5. 4 - 1st derivative ESR spectra

H

65G

(a) ~ -irradiated wool premodified by reduction and iodoa­ cetylation.

(b) Per sulphate oxidized wool premodified by reduction and iodacetylation. FIG. 5.5 - 1st derivative ESR spectra

H

65G

(a) 15 -irradiated wool containing lanthionine cross-links.

(b) Per sulphate oxidized wool containing lanthionine cross-links. FIG. 5. 6 - 1 st derivative ESR spectra

H

65G

(a) c5 -irradiated wool.

(b) Per sulphate oxidized wool. - 145 -

The chemical pre-modifications do not prevent the formation of the free radicals but it is evident that the spectra differ from that of unmodified wool. The spectra of these modified wools have one feature in common and that is the diminution and/or removal of the low frequency component of the spectrum of normal wool. Since these premodifications disrupt the disulphide bond this provides additional evidence to suggest that this spectral component is associated with the disulphide bond of the cystyl residues. However, such an interpretation requires that the chemical modifications carried out on wool are completely selective for the disulphide group and, as has been already pointed out, this is not the case for at least two of these modifications. From the fact that peracetic acid oxidation of wool prevents subsequent participation of the sulphur from the usual free radical formation reactions, and because of its generally accepted specificity toward the disulphide bond, it is perhaps significant that this modification has succeeded in removing the low frequency spectral component from the spectrum of wool without much alteration to the remaining spectral feature. The more general nature of the attack on wool, occurring with the other pre-modifications, may be responsible for the changes occurring in the latter feature of the ESR spectra. On the basis of this evidence, it may be concluded that - 146 - the ESR spectrum of free radicals formed in wool is not a result of one contributing structure as previously thought. It appears to be the resultant of several overlapping structures, one of which seems to be associated with the disulphide bond. (b) Modificatjon of the terminal and free amino groups in wool. The free amino groups in wool were blocked by means of the following modifications: (i) Acetylation. (ii) Reaction with 2,4-dinitrofluorobenzene {DNFB). The acetylation of the free amino groups in wool may be represented by the following equation:

CH3-c~ '\ H+ 1o ---=-+ R-cH2-NH-cocH3 + cH3-cooH CH3-CO

There is a possibility that the alcoholic hydroxyl groups of serine and threonine as well as the phenolic hydroxyl group of tyrosine may be modified by this treatment. The reaction of DNFB with wool is not specific for the free and terminal amino groups since it is known that DNFB reacts with the 0(.-imino group of histidine, the phenolic hydroxyl group of tyrosine and the thiol groups of cyste1ne145• Figures 5.7, 5.8 illustrate the ESR spectra of these modified wools after 1S" -ray irradiation and persulphate oxidation. Similarities are again discernible in the spectra FIG. 5. 7 - 1st derivative ESR spectra

H )

65G

(a) ~ -irradiated acetylated wool.

(b) Persulphate oxidized acetylated wool. FIG. 5. 8 - 1st derivative ESR spectra

65G )

(a) 15 -irradiated wool pretreated with 2, 4 dinitrofluorobenzene.

(b) Persulphate oxidized wool pretreated with 2, 4 dinitro­ fluorobenzene. - 147 - of any one type of wool irrespective of the mode of free radical formation employed. The results obtained have not elucidated the function of the free and terminal amino groups of wool in free radical formation. Op the one hand, blocking of this group by acetylation yields a spectrum similar to that of unmodified wool, suggesting that this grouping is not involved in free radical formation. DNFB modification of wool, on the other hand, has completely altered the size and the shape of the spectrum from that of unmodified wool. However, since it has been found that the ESR spectrum of <5-ray irradiated DNFB modified silk (shown in Figure 5.9) appears to be identical in structure with that of lS -ray irradiated and oxidised DNFB-wool, whereas the ESR spectra of oxidized or irradiated unmodified silk (shown in Figure 6.10 (a) and (b) ) are different from the corresponding spectra of wool, as might be expected from the difference in chemical composition of these proteins, the free radicals produced in DNFB modified proteins are probably associated with the dinitrophenyl residues. (c) Modification of the combined tyrosine. The aromatic ring of the combined tyrosine was modified in the following ways: (1) Substitution in the ring by iodination. (11) Methylation of the phenolic hydroxyl group with diazanethane. FIG. 5. 9 - 1st derivative ESR spectrum of '?S' -irradiated silk premodified with 2, 4 dinitrofluorobenzene.

H )

65G (; ) FIG. 5. 10 - 1st derivative ESR spectra

65G )

(a) O -irradiated silk.

(b) Per sulphate oxidized silk. - 148 -

Iodination of the combined tyrosine in wool forms 3,6- diiodotyrosyl residues and this can be illustrated by the following equation, I R-CH2 -0-oH + 212 ~ R-CH2---aOH + 2HI

The reaction of iodine with wool has been shown to be capable of also oxidizing disulphide bonda146• The modification of the phenolic hydroxyl groups in canbined tyrosine by treatment with diazomethane can be shown as follows:

Diazomethane is known to be capable of methylating the free carbo.xyl groups which occur in wool in the side chains of aspartic and glutamic acids. The spectra of these modified wools are shown in Figures

5.11 (a) and (b), 5.12 and show significant differences from the spectrum of the control. The spectra of iodinated-wool show variations only in the high-frequency spectral component; the low frequency component appears to be unaltered by the modification. In the case of diazomethanetreated wool, the low frequency component, relative to the remaining component, seems to be slightly reduced in intensity in comparison with the control. Since the former component is generally associated with the disulphide bond, the reason for this effect FIG. 5. 11 - 1st derivative ESR spectra

65G )

(a) O -irradiated wool premodifie with iodine.

(b) Per sulphate oxidized wool premodified by iodine. FIG. 5. 12 - 1st derivative ESR spectrum of lS -irradiated wool pretreated with diazomethane

H >

65G ) - 149 - ie obscure as aiazomethane is not known to modify thie group. The fact that iodinated wool exhibits changes in the high frequency spectral component indicates that tyrosine is possibly a contributor to this part of the ESR spectrum. Iodination of .the tyrosine will block the two ortho-positione of the aromatic ring and these are normally the sites of highest electron density due to the ortho-para directing effect of the phenolic hydroxyl group. In the case of diazomethane treated wool the ortho-positions are not blocked although the ortho-para directing effect of the phenolic group is now replaced by that of the methoxy group and therefore, if tyrosine is a location for the unpaired electrons in keratin, the diazomethane treatment should have little effect on the spectral hyperfine structure. (a) Modification of the free carboxyl groups in wool. The free and terminal carboxyl groups in wool were modified by selective esterification with methanol and this reaction may be represented by the following equation,

H+

The ESR spectra of this wool formed on subsequent free radical formation are shown in Figures 5.13 (a) and (b). It can be seen that the spectra are similar to those of the controls, the or,ly significant difference being a slight variation in the high-frequency component. FIG. 5. 13 - 1 st derivative ESR spectra

H >-

65G (

(a) Persulphate oxidized methylated wool.

(b) lS -irradiated methylated wool. - 150 -

The above results indicate that ESR spectral evidence in conjunction with prior chemical modification of wool can yield more informative data concerning the possible unpaired electron envirorunents than is possible from the spectrum of unmodified wool alone. Even so, interpretation of the results is difficult since in most cases the exact nature of the modified wool is not known. The results obtained, however, have indicated that the ESR spectrum of wool is not caused by a single unpaired electron environment but is in fact made up of several environments which possibly involve the cystine disulphide bond and the phenolic ring of tyrosine. Perhaps the main significance of these results lies in the similarity of the ESR spectra of the various chemically pre-modified wools irrespective of the mode of free radical generation, implying that oxidation and high-energy irradiation induce the same effects in proteins. The ESR results, however, provide only indirect evidence concerning the free 1·adical environments in oxidized or irradiated wool and therefore, it is necessary to suppliment this from some alternative source such as direct chemical analyses of the treated fibres.

Chemical Analyses of Irradiated and Oxidized Wool The effect of high-energy radiation on wool keratin has been investigated by a number of workers. The amino acid residues mainly affected are tryptophyl, tyrosyl alld cysty1147 - 151 - although Kenny148 has found that with high doses lysyl and prolyl residues are also degraded. Since the chemical modifications occurring when free radicals ar·e formed in wool, subjected to the combined effect of sodium pers~lphate and heat, have not been the subject of any detailed investigation, it was decided to carry out a total amino acid analysis on wool treated in this fashion. The wool was treated at room temperature for ten minutes with a solution of sodium persulphate (2%) in sulphuric acid (O.OlN), containing a small amount of the non-ionic wetting agent Lissapol N. The wool was then centrifuged to remove excess oxidant solution and next placed in an oven and heated to 110°c ! 2°c for ten minutes in an atmosphere of dry nitrogen. The analysis was carried out by c.s.I.R.o., Division of Protein Chemistry, Parkville N.2, Victoria and is shown in Table 6.1. The results indicate that the ccmbined cystine and tyrosine are modified by persulphate oxidation of wool. The formation of dichlorotyrosine is clearly caused by the presence of sane residual oxidant oxidizing the chloriue ions to chlorine which then substitutes the aromatic ring78• However, there does appear to be a reel loss of tyrosine from the initial oxidation149• The differences in the values of the other amino acids are not thought to be significant. Needles36 has recently investigated the chemical changes produced by the action of persulphate on wool and has found - 152 -

TABLE 5.1 Amino Acid Analysis of Wool Oxidized by Sodium Persulphate Amino acid nitrogen as a percentage of total nitrogen applied Wool Amino Acid. Control Treated lysine 4. 34 4. 02 histidine 1.86 2. 28 annnonia 9.18 10. 31 argenine 18.56 17.56 aspartic acid 4.54 4. 39 threonine 4. 33 4.11 serine 6.81 6. 11 glutamic acid 8.15 0.02 praline 4. 95 4. 68 glycine 6.08 5. 92 alanine 3. 82 3. 73 cystine/2 7. 32 6. 78 valine 4.13 4. 20 methionine o. 31 o. 29 isoleucine 2. 27 2. 29 leucine 5. 37 5. 25 tyrosine 2. 58 2. 01 phenylalanine 1.96 1.81 cysteic acid trace o. 58

dichlorotyroeine 0 o. 76 - 153 -

that, apart from cystine and tyrosine, losses in histidine, proline and methionine also occur. The results exhibited in Table 5.1 show no significant modification of the latter three amino acids but this discrepancy may be associated with the more drastic conditions employed by Needles. The increase in annnonia-nitrogen has also been observed by Needles and this is attributed to attack on the peptide-chain between the CX-carbon and nitrogen atoms. Graham and Stathami7 have reported that persulphate modifies the tryptophyl residues in wool but Needles36 has not carried out any analyses for this residue. It was demonstrated in the previous chapter that the oxidation of the soluble proteins casein and lysozyme by persulphate is restricted primarily to the tryptophyl residues and therefore, it was decided to measure the tryptophan content of wool oxidized by per sulphate. This determination was carried out on wool treated for various times in solutions of sodium persulphate of various concentrations (liquor to wool ratio 50:1) after which the wool was centrifuged and then heated for ten minutes at 110°0 in an atmosphere of nitrogen. The results, obtained by the analytical procedure described in Appendix A2, are shown in Table 5.2. It is evident that, apart from combined tyrosine and cystine, tryptophan is easily degraded by the action of persulphate on wool and it is likely, therefore, that these - 154 -

TABLE 5. 2

'l'he Modification of Tryptophan in Wool Oxidized by Sodium Persulphate Concentration of Treatment Time in Tryptophan Sodium Persulphate Oxidant

~ (minutes) 00. 0

0.1 10 0.46 o. 2 10 0.45 o. 75 30 0.35 residues provide some of the locations of the free radicals generated in keratin. Conf'irmation of this by the ESR method was not possible as no satisfactory method of specifically, chemically blocking the tryptophan residues could be found. In fact the above treatment with persulphate seems as successful as any method yet tried for the specific removal of tryptophan from wool.

Conclusions The formation of free radicals in wool may be considered to represent a partial oxidation of this protein and this will proceed to completion if oxygen or air is present in the system. Two possibilities are envisaged; (1) The free radicals are converted into oxidation products (modified amino acid residues not normally present in WllDodified wool). - 166 -

(11) Some of the amino acids formed by the ultimate oxidation of the free radicals may in fact be identical with amino acid residues originally present. For example, if the cystyl residue in wool undergoes carbon-sulphur bond fission as shown,

R-CH2-S-S-CH2-R ~ R-CH2-s-s· + (I) it is conceivable that the free radical (II) may form either alanine or serine, after contact with air and subsequent acid hydrolysis, while the free radical (I) will afford products not normally present. Under these circwnstances the forma- tion of alanine or serine by oxidation may mask the fact that alanine or serine are themselves degraded in the system. Because of this possible mechanism, chemical analyses of amino acids originally present in wool before and after free radical formation may give misleading information concerning the environments of unpaired electrons in wool. More precise information concerning the sites of unpaired electrons in treated wool should be obtained by the identifi- cation of the products. Forbes and coworkers161 have used both electrophoretic and paper chromatographic techniques for separating and identifying products formed by ultraviolet and -irradiation of the naturally occurring, sulphur-containing amino acids. These workers have found that the irradiation of any of these amino acids in solution yields a large variety - 156 - of products, the nature of which depends on various parameters of the irradiation system. It is clear that if a simple amino acid forms such a large number of products upon irradiation, then it would be expected that. the number of products formed by irradiation of a complex protein such as wool would be much higher. Even if it were possible to separate and identify all products formed on "l5-irradiation or oxidation of wool, the result may still not indicate the sites of unpaired electrons in the original polypeptide, for there is a distinct possibility that modifications to the original oxidation products occur during subsequent treatments necessary for their isolation and determination. - 157 -

SUMMARY

The preceding investigation was undertaken to obtain information concerning the chemical processes occurring when wool is treated with oxidizing agents. Because of the inherent insolubility of wool keratin and the extremely complex histology of the fibre, chemical analyses of the oxidized fibre, in general, can only be attempted after hydrolysis of the protein into its constituent amino acids has been achieved. In view of the fact that hydrolysis of oxidized wool may result in subsequent chemical changes, it was decided to investigate instead the oxidation of some model compounds related to wool, such as the free Ol-amino acid constituents of wool and certain soluble proteins. This approach enables a number of physico-chemical techniques to be employed to study the oxidation processes occurring in these model compounds thereby obtaining some insight into the possible reactions occurring with wool. The potentiometric investigation has yielded useful data concerning two facets of the oxidation of the free amino acid components of wool. First, the method gives an indication of the susceptibility to oxidation of certain amino acid canponents and secondly, it is sometimes possible to deduce the probable oxidation products from the location of the potentianetric end-point. During the potentiometric titration, the rate at - 158 - which the potential, corresponding to the oxidant, decreases gives some indication of the rate of oxidation of the amino acid and this has been used to demonstrate that of the amino acid canponents of wool, only cystine, cysteine, methionine, tyrosine and.tryptophan are oxidized rapidly under the conditions employed. Both potassium permanganate and chlorine appear to react rapidly with cysteine, methionine and tryptophan but they differ markedly in their attack on cystine and tyrosine. Thus, chlorine has been found to react rapidly with cystine but only slowly with tyrosine, while with permanganate the reactivities appear to be reversed, tyrosine being oxidized quickly whereas cystine reacts only slowly. The presence of high concentrations of sodium chloride has been found to influence the permanganate oxidation of tyrosine and tryptophan, and it appears that the oxidation does not reach the level attained when salt is absent. The presence of the salt has been found to accelerate greatly the oxidation of cystine by permanganate but the actual function of the salt has not been elucidated. It has been shown that large concentrations of the halide ions, chloride or bromide, cause the rapid disruption of the manganese (III) complex with the reduction of the manganese (III) to manganese (II), whereas the other salts employed, such as sodium sulphate or sodium nitrate, were ineffective in this - 159 - regard. This result indicates that the presence of large concentrations of halide ions may function by initially increasing the rate of oxidation of the cystyl residues in addition to restricting the reaction to the surface of the fibre as has been proposed by other workers. The oxidation of the two soluble proteins, insulin and lysozyme, and of mixtures of amino acids corresponding to their concentrations in these proteins was also studied by the potentiometric technique. It was found that the potentiometric end-points, for the permanganate oxidation of insulin and its corresponding amino acid mixture, agree very closely to that required for the specific oxidation of the tyrosyl component, based on the results obtained for the oxidation of free tyrosine by permanganate. On this basis and from the fact that tyrosine is oxidized more rapidly than cystine by perman­ ganate, it was thought that this result signifies that the tyrosyl residues of insulin are oxidized preferentially by permanganate. For insulin oxidized by chlorine, it was found that the potentiometric end-point for the protein was somewhat greater than that for its corresponding amino acid mixture, which yielded the same end-point calculated for the oxidation of the cystine content alone. This result was interpreted as indicating that chlorine oxidizes the cystyl residues preferentially from mixtures of the free amino acids but in the case of the protein other groups are also attacked. By - 160 - employing a similar argument, it appears that the oxidation of lysozyme by permanganate occurs by preferential modification of its tryptophyl residues whereas its oxidation by chlorine is somewhat less specific. In order-to investigate further the possibility that preferential oxidation of tyrosine or cystine occurs from mixtures of these amino acids, it was decided to employ a polarographic technique. It was found that, whereas chlorine and permanganate both cause a lowering in the height of the polarographic wave of cystine, the inclusion of tyrosine modifies this behaviour markedly. Thu~ in the presence of tyrosine it is found that permanganate does not lower the height of the polarographic wave of cystine and, therefore, it may be concluded that the cystine is unaffected by permanganate which, consequently, must react preferentially with the tyrosine. The presence of tyrosine is found not to alter the behaviour of chlorine on the polarographic wave of cystine and from this it may be deduced that the chlorine reacts preferentially with the cystine. This difference in behaviour was utilized in determining whether the oxidation of cystine in the presence of sodium chloride by permanganate proceeded by a chlorination reaction. It was found, by including tyrosine in the system containing cystine and salt, that the permanganate produced virtually no effect on the height of the polarographic wave of cystine and, therefore, - 161 - the permanganate-salt oxidation resembles that of a permanganate oxidation and not that of a chlorination. The polarographic method, however, is applicable only to the free amino acids and in order to determine whether this preferential attack may also occur fo~ these amino acids combined in proteins, it was decided to employ the technique of ultraviolet difference spectroscopy. The oxidation of the free amino acids tyrosine, tryptophan and cystine generally results in the formation of difference spectra which, except for the case of cystine, possess features that characterize the particular amino acid being examined. The difference spectra generated by the oxidation of tryptophan are characterized by an "abstraction" pattern of negative optical density arising from the loss of part of the original tryptophan in the oxidized sample. From additional evidence obtained by chromatography and ultraviolet absorption spectroscopy, it appears that the "abstraction" feature of negative optical density results from the disruption of the indole rine into products such as kynurenine, anthranilic and 3-hyd.roxyanthranilic acid. The difference spectra generated by the oxidation of tyrosine also possess an "abstraction" spectra arising from loss of part of the original tyrosine in the oxidized sample. The ultraviolet difference spectral investigation of the oxidation of the soluble proteins has yielded useful information - 162 - concerning the susceptibilities of the aromatic amino acid residues, in particular tyrosyl and tryptophyl, toward oxidation by a variety of oxidizing agents. A comparison of the difference spectra formed by the oxidation of the soluble proteins and mixtures of tyrosine, tryptophan and cystine, in the same proportions as their occurrences in these proteins, has shown that the oxidation of the proteins may, as a first approximation, be accounted for in terms of these three amino acids. The oxidation of insulin has provided useful data con­ cerning the oxidation of tyrosyl and cystyl residues in proteins, since insulin possesses these residues but does not contain tryptophan, cysteine nor methionine. The oxidation of insulin by potassium permanganate produces a difference spectrum which can be accounted for entirely by the oxidation of the tyrosyl residue. Furthermore, this difference spectrum was found to be identical, except for the slight bathochromic shifts resulting from the incorporation of the amino acids into polypeptide chains, to that produced by the oxidation of the free amino acids occurring in the same proportions as their concentrations in insulin. It is thought, therefore, that the oxidation of insulin by permanganate occurs preferent­ ially at the tyrosyl residues. Oxidation of insulin by chlorine produces rather feature­ less difference spectra showing a gradual increase in optical - 163 - densities below 340 mp.. These spectra do not appear to have any significant features which could be ascribed to the oxidation of the tyrosyl residues and, therefore, they are thought to arise from the oxidative modification of cystyl residues and possibly other groups in the protein with little, if any attack on the tyrosyl residues. These results indicate that the preferential oxidation of tyrosine or cystine, from their mixtures by potassium permanganate and chlorine respectively, as noted previously, appears to occur in the combined state as well. This does not necessarily imply that this behaviour occurs during the oxidation of fibrous proteins, such as wool and silk, by these oxidants since the reaction in this latter case is a heterogeneous one. In the case of the oxidation of insulin by sodium persulphate, the uifference spectral evidence suggests that preferential attack of the tyrosyl residues is taking place, while there is some evidence to suggest that modification of this residue also occurs with peracetic acid. The protein casein provides information about the susceptibility of tyrosyl and tryptophyl residues toward oxidizing agents since this protein has an extremely low cystine content. Thus, the difference spectra obtained by the oxidation of casein with potassium permanganate show that the oxidation occurs mainly at the tryptophyl residue, although there is some evidence that the tyroeyl residues are also - 164 -

affected. In the case of the reaction between chlorine and casein, the difference spectral evidence indicates that the oxidation proceeds initially by the preferential oxidation of the tryptophyl residues after which oxidation of the tyrosyl residues occurs, whereas the oxidation by sodium persulphate yields difference spectra which can only be attributed to oxidative modification of the tryptophyl residues, implying that persulphate attacks this residue specifically. In the case of the peracids there is some indication that peracetic acid modifies the tryptophyl residues of casein but with permonosulphuric acid there is little evidence for modifica­ tion of either tyrosyl or tryptophyl residues under the conditions employed. The protein lysozyme provides an extremely useful model for the study of the oxidation of cystyl, tryptophyl and tyrosyl residues. The results of the difference spectral analyses indicate that the oxidants, potassium permanganate, chlorine ann souium persulphate oxidize the tryptophyl residues of this protein preferentially although with chlorine it appears that the cystyl residues are affected concurrently. The difference spectra produced by the oxidation of lysozyme ~ith peracetic acid exhibit some features which may be ascribed to the modification of the tryptophyl residue, but there is f-'.Ome eviderice that other resjclues are being oxidi1ed. In the cose of permonosulphuric acid. oxidized lysozyme the1·e is - 165 - some evidence of tryptophyl modification but the difference spectra exhibit extremely low absorptivities. In the light of the known reactivity between cystine and the peracids it would appear that the cystyl residues of lysozyme are under­ going oxidatipn in this case. The examination of the oxidation of the fibrous proteins silk and wool by the technique of ultraviolet difference spectroscopy was attempted. In the case of silk, which contains tyrosine but virtually no tryptophan nor cystine, it was possible to dissolve silk by an acid hydrolysis employing sulphuric acid. and the diff'erence spectra obtained inuicate that both permanganate and chlorine react with the tyrosyl residues of this protein. Because of the inherent insolubility of wool, associated with the extremely complex histology of the fibre, it was found that dissolution without extensive chemical modification was not possible and, therefore, no conclusions concerning the preferential oxidation of the tyrosyl, tryptophyl or cystyl residues of wool was possible. In order to obtain information concerning the oxidation of wool keratin, a study of the free radicals generated by partial oxidation was undertaken. It was found that of all the oxidizing agents employed in this investigation, only sodiwn persulphate was capable of producing sufficiently high concentrations of free radicals to be detectable by ESR spect1·oscopy and, therefore, the investigation was limited to - 166 - the use of this oxidant. The hyperfine structure of the ESR spectrum of persulphate oxidized wool is very similar to that of ~-ray irrauiated wool and, since the hyperfine structure is intimately related to the environments of the free radicals, it appears that the modifications occurring in either case are similar. To determine the free radical environments in wool it was necessary to modify chemically various groups in wool before subjecting it to free radical formation. In this way it is possible to determine whether modification of a particular group in wool causes an alteration in the hyperfine structure of wool. It was found that pre-modification of the disulphide bonds of the cystyl residues always resulted in the removal of the low frequency component of the ESR hyperfine structures of oxidized and irradiated unmodified wool and from this it would appear that the disulphide bond is one of the environments for free radicals in wool. Modification of the free and terminal amino groups in wool by acetylation was found to have little effect on the overall hyperfine structure, yet the blocking of those groups with dinitrofluorobenzene was found to alter the ESR hyperfine structure of wool considerably. Treatment of silk with this reagent was found to produce the same hyperfine structure on radical formation and from this it was deduced that the free radicals were associated with the dinitropheeyl groups introduced into these proteins. - 167 -

Blocking of the two ortho-positions of the phenolic ring of tyrosine by iodination was found to modify the high­ frequency component of the ESR spectrum of wool, thereby indicating that the tyrosyl residues probably contribute to this part of .the ESR spectrum. Methylation of the phenolic hydroxyl group, on the other hand, whereby the two ortho positions are not significantly affected was found to have little affect on the high frequency component of the ESR spectrum of wool. The free carboxyl groups of aspartic and glutamic acids appear not to be involved in free radical formation since esterification of these groups was found to have no effect on the shape of the ESR spectrum of wool. The possibility that the tryptophyl residues of wool are another free radical environment could not be confirmed by ESR spectroscopy. However, on the basis of direct chemical analyses and on the work carried out on the soluble proteins casein and lysozyme it is likely that combined tryptophan is an additional location for free radicals in wool. The preceding investigation has shown, therefore, that the oxidation of proteins appears to proceed in preferential stages involvi1ig combined cystine, tyrosine and tryptophan anu depending on the particular oxidizing agent employed. Thus, potassium permanganate, sodium persulphate and chlorine are found to attack preferentially tryptophyl residues, whenever these are present in the protein, although with - 168 - chlorine it appears that oxidation of the cystyl residues occurs simultaneously. In the case of proteins lacking tryptophan, it seems that permanganate and persulphate oxidize tyroeyl residues specifically whereas chlorine attacks combined cyetine preferentially. In all cases, oxidative attack on proteins by the peracids appears to be mainly with the cystyl residues with very little attack on the aromatic amino acid residues. It is interesting to relate these results, assuming similar reactions occur with wool, to a general theory of oxidative shrinkproofing of wool, assuming of course, that oxidative shrinkproofing eventuates from chemical modification of the fibre at the molecular level. Thus, the oxidation of combined cystine appears to be a necessary reaction for oxidative shrinkproofing of wool. This view is supported by the fact that oxidizing agents such as chlorine and potassium permanganate/sodium chloride are extremely effective shrinkproofing agents and have been shown to react rapidly with cystine, whereas potassium permanganate alone is rather ineffective in this regard and it has been found to oxidize preferentially the aromatic residues, tyrosyl and tryptophyl, and to react only slowly with cystine. Disulphide bond oxidation, however, is not the only necessary reaction for oxidative shrinkproofing, since peracetic acid attacks this group in wool preferentially without achieving significant - 169 - ahrinkproofing. In this regard it would appear that the oxidation of the aromatic residues, tyrosyl and tryptophyl, is incidental to the mechanism of oxidative shrinkproofing, since sodium persulphate and potassium permanganate, which readily attac~ these residues, are rather ineffective shrinkproofing agents. Possibly main chain degradation is a necessary requirement, in addition to combined cystine oxidation, for the oxidative shrinkproofing of wool but this aspect was not considered in the present investigation. A detailed study of the oxidative degradation of peptide main chains in model compounds and proteins is necessary, therefore, before any definite conclusions may be drawn concerning the involvement of main chain degradation in the oxidative shrinkproofing of wool. - 170 -

APPENDIX

Al. The Polarographic Determination or Cystine and Cysteine The method of Ben!Mek was employed for the polarographic determination of cystine and cysteine115• The polarographic cell employed was constructed by Laboratory Supply Pty. Ltd., (Sydney) and is illustrated by Figure A. 1. The cell is prepared in the following manner: Sufficient mercury is introduced to chamber B through tap C to cover the overflow outlet E. Saturated sodium sulphate in sulphuric acid (0.lN) is next added to fill chamber B with tap D closed. The reservoir R is next filled with the saturated sodium sulphate and with tap Cleft opened, tap D is opened so that the solution fills the stop-cock opening

of tap D which is then closed. A:ny excess saturated sodium sulphate solution in the side-arm of chamber A is removed by means of a pipette fitted with a length of flexible polyethylene tubing. The side-arm is rinsed out several times with distilled water which is also subsequently removed and tap C is then closed.

The solution to be analysed is introduced into chamber A with both taps D and F closed. Gelatin (0.20 ml. of 0.5% solution) is added to suppress the formation of any maxima

during the actual polarographic determination. Chamber A is sealed with a rubber stopper containing the capillary FIG. Al. Diagram of polarographic cell employed

G

E - 171 - through which the mercury flows. Oxygen-free nitrogen is then passed for at least ten minutes through the solution by means of inlet G to remove dissolved oxygen from the solution. The nitrogen flow is halted at the end of this period, tap D is opened and.a current-voltage curve is recorded from zero to -1.6 volts by employing a Cambridge pen recording polaro-

~~~ A calibration curve showing the relationship between the wave height and the concentration of cystine is presented in Figure A.2, from which it is evident that the wave height is directly proportional to the concentration of cystine solution over the concentration range studied. FIG. A2. - Polarographic calibration curve for cystine wave­ height versus concentration.

Curves: (i) 0. 20 ml.; (ii) 0. 15 ml.; (iii) 0. 10 ml.; (iv) 0. 05 ml.; (v) 0. 00 ml., of cystine solution ( 1 mg/ml). Curves (i) - (v) from O volts. 200 mv/abscissa unit. - 172 -

A2. The Determination of Tryptophan in Wool Mazingue and van Overbeke150 showed that tryptophan in wool gives a blue colour with P -dimethyl amino benzaldehyde and furthermore, that under the conditions employed, the reagent was specific for this amino acid. In order to complete the acid hydrolysis of wool without destruction of the tryptophan, the hydrolysis was carried out at room temperature in the presence of the colour forming reagent. Dry wool (0.1 gm.) was immersed in 25 mls. of concentrated hydrochloric acid to which was added immediately 1 ml. of p -dimethyl amino benzaldehyde (a 5% solution in 10% sulphuric acid). A blue colour developed within two or three hours and continued to develop as the wool gradually dissolved. 'l'his colouration reached a maximum after 7 or 8 days, that is at the time of complete hydrolysis of the wool and remained stable for several days following. The solution was diluted to 50 mls. and the optical density was then read at 6050 R. Calibration was carried out by using tryptophan solutions of known concentrations and from the optical density value obtained for a known weight of wool, the tryptophan content was determined. - 173 -

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